Delivery across cell plasma membranes

ABSTRACT

Delivering a payload across a plasma membrane of a cell includes providing a population of cells and contacting the population of cells with a volume of an aqueous solution. The aqueous solution includes the payload and alcohol content greater than 5 percent concentration. The volume of the aqueous solution may be a function of exposed surface area of the population of cells, or may be a function of a number of cells in the population of cells. Related compositions, apparatus, systems, techniques, and articles are also described.

RELATED APPLICATION

This application is a continuation of and claims the benefit under 35U.S.C. § 120 of U.S. patent application Ser. No. 16/841,236, filed onApr. 6, 2020, presently pending, which is a continuation of U.S. patentapplication Ser. No. 15/521,192, filed on Apr. 21, 2017, issued as U.S.Pat. No. 10,612,042, which is a national stage application filed under35 U.S.C. § 371, of International Patent Application No.PCT/US2015/057247 filed Oct. 23, 2015, which claims the benefit of andpriority to British application no. 1419013.6 filed Oct. 24, 2014, toBritish application no. 1419012.8 filed Oct. 24, 2014, and to Britishapplication no. 1419011.0 filed Oct. 24, 2014, the entire contents ofeach of which are hereby expressly incorporated by reference herein.

TECHNICAL FIELD

The present subject matter relates to delivering agents cell plasmamembranes. The present subject matter may include, for example,delivering molecular, biological and pharmacological therapeutic agentsto a target site, such as a cell, tissue, or organ.

BACKGROUND

Despite technical advances in some areas, delivery of certain moleculesinto cells remains a challenge because of factors such as size or chargeof the molecule. A plasma or cell membrane is a semi-permeablebiological membrane, which acts as a selective barrier, regulating thechemical composition of a cell. Therefore, only certain molecules cantranslocate across the plasma membrane by passive diffusion into a cell.Small, hydrophobic molecules (such as O₂, CO₂ and N₂) and small,uncharged polar molecules (such as H₂O and glycerol) can passivelydiffuse across a plasma membrane. Larger, uncharged polar molecules(such as amino acids, glucose, and nucleotides) and ions (such as H⁺,Na⁺, K⁺ and Cl⁻) cannot passively diffuse into a cell.

SUMMARY

The invention is based on the surprising discovery that compounds ormixtures of compounds (compositions) are delivered into the cytoplasm ofeukaryotic cells by contacting the cells with a solution containing acompound(s) to be delivered (e.g., payload) and an agent that reversiblypermeates or dissolves a cell membrane. Preferably, the solution isdelivered to the cells in the form of a spray, e.g., of five aqueousparticles. For example, the cells are coated with the spray but notsoaked or submersed in the delivery compound-containing solution.Exemplary agents that permeate or dissolve a eukaryotic cell membraneinclude alcohols and detergents such as ethanol and Triton X-100,respectively. Other exemplary detergents, e.g., surfactants includepolysorbate 20 (e.g., Tween 20),3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), sodium dodecyl sulfate (SDS), and octyl glucoside.

An example of conditions to achieve a coating of a population of coatedcells include delivery of a fine particle spray, e.g., the conditionsexclude dropping or pipetting a bolus volume of solution on the cellssuch that a substantial population of the cells are soaked or submergedby the volume of fluid. Thus, the mist or spray comprises a ratio ofvolume of fluid to cell volume. Alternatively, the conditions comprise aratio of volume of mist or spray to exposed cell area, e.g., area ofcell membrane that is exposed when the cells exist as a confluent orsubstantially confluent layer on a substantially flat surface such asthe bottom of a tissue culture vessel, e.g., a well of a tissue cultureplate, e.g., a microtiter tissue culture plate.

Accordingly, there is a need to provide a vector-free e.g., viralvector-free, approach for delivering biologically relevant payloads,e.g., compounds or compositions, across a plasma membrane and intocells. “Cargo” or “payload” are terms used to describe a compound, orcomposition that is delivered via an aqueous solution across a cellplasma membrane and into the interior of a cell.

In an aspect, delivering a payload across a plasma membrane of a cellincludes providing a population of cells and contacting the populationof cells with a volume of an aqueous solution. The aqueous solutionincludes the payload and an alcohol content greater than 5 percentconcentration. The volume of the aqueous solution may be a function ofexposed surface area of the population of cells, or may be a function ofa number of cells in the population of cells.

In another aspect, a composition for delivering a payload across aplasma membrane of a cell includes an aqueous solution including thepayload, an alcohol at greater than 5 percent concentration, less than46 mM salt, less than 121 mM sugar, and less than 19 mM buffering agent.For example, the alcohol, e.g., ethanol, concentration does not exceed50%.

One or more of the following features can be included in any feasiblecombination. The volume of solution to be delivered to the cells is aplurality of units, e.g., a spray, e.g., a plurality of droplets onaqueous particles. The volume is described relative to an individualcell or relative to the exposed surface area of a confluent orsubstantially confluent (e.g., at least 75%, at least 80% confluent,e.g., 85%, 90%, 95%, 97%, 98%, 100%) cell population. For example, thevolume can be between 6.0×10⁻⁷ microliter per cell and 7.4×10⁻⁴microliter per cell. The volume is between 4.9×10⁻⁶ microliter per celland 2.2×10⁻³ microliter per cell. The volume can be between 9.3×10⁻⁶microliter per cell and 2.8×10⁻⁵ microliter per cell. The volume can beabout 1.9×10⁻⁵ microliters per cell, and about is within 10 percent. Thevolume is between 6.0×10⁻⁷ microliter per cell and 2.2×10⁻³ microliterper cell. The volume can be between 2.6×10⁻⁹ microliter per squaremicrometer of exposed surface area and 1.1×10⁻⁶ microliter per squaremicrometer of exposed surface area. The volume can be between 5.3×10⁻⁸microliter per square micrometer of exposed surface area and 1.6×10⁻⁷microliter per square micrometer of exposed surface area. The volume canbe about 1.1×10⁻⁷ microliter per square micrometer of exposed surfacearea. About can be within 10 percent.

Confluency of cells refers to cells in contact with one another on asurface. For example, it can be expressed as an estimated (or counted)percentage, e.g., 10% confluency means that 10% of the surface, e.g., ofa tissue culture vessel, is covered with cells, 100% means that it isentirely covered. For example, adherent cells grow two dimensionally onthe surface of a tissue culture well, plate or flask. Non-adherent cellscan be spun down, pulled down by a vacuum, or tissue culture mediumaspiration off the top of the cell population, or removed by aspirationor vacuum removal from the bottom of the vessel.

Contacting the population of cells with the volume of aqueous solutioncan be performed by gas propelling the aqueous solution to form a spray.The gas can include nitrogen, ambient air, or an inert gas. The spraycan include discrete units of volume ranging in size from, 1 nm to 100μm, e.g., 30-100 μm in diameter. The spray includes discrete units ofvolume with a diameter of about 30-50 μm. A total volume of aqueoussolution of 20 μl can be delivered in a spray to a cell-occupied area ofabout 1.9 cm², e.g., one well of a 24-well culture plate. A total volumeof aqueous solution of 10 μl is delivered to a cell-occupied area ofabout 0.95 cm², e.g., one well of a 48-well culture plate. Typically,the aqueous solution includes a payload to be delivered across a cellmembrane and into cell, and the second volume is a buffer or culturemedium that does not contain the payload. Alternatively, the secondvolume (buffer or media) can also contain payload. In some embodiments,the aqueous solution includes a payload and an alcohol, and the secondvolume does not contain alcohol (and optionally does not containpayload). The population of cells can be in contact with said aqueoussolution for 0.1-10 minutes prior to adding a second volume of buffer orculture medium to submerse or suspend said population of cells. Thebuffer or culture medium can be phosphate buffered saline (PBS). Thepopulation of cells can be in contact with the aqueous solution for 2seconds to 5 minutes prior to adding a second volume of buffer orculture medium to submerse or suspend the population of cells. Thepopulation of cells can be in contact with the aqueous solution, e.g.,containing the payload, for 30 seconds to 2 minutes prior to adding asecond volume of buffer or culture medium, e.g., without the payload, tosubmerse or suspend the population of cells. The population of cells canbe in contact with a spray for about 1-2 minutes prior to adding thesecond volume of buffer or culture medium to submerse or suspend thepopulation of cells. During the time between spraying of cells andaddition of buffer or culture medium, the cells remain hydrated by thelayer of moisture from the spray volume.

The aqueous solution can include an ethanol concentration of 5 to 30%.The aqueous solution can include one or more of 75 to 98% H₂O, 2 to 45%ethanol, 6 to 91 mM sucrose, 2 to 35 mM KCl, 2 to 35 mM ammoniumacetate, and 1 to 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) (HEPES).

The population of cells can include adherent cells or non-adherentcells. The adherent cells can include at least one of primarymesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells,neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinesehamster ovary (CHO) cells, and human embryonic kidney (HEK) cells orimmortalized cells, such as cell lines. The population of cells caninclude non-adherent cells. The non-adherent cells can include at leastone of primary hematopoietic stem cell (HSC), T cells, natural killer(NK) cells, cytokine-induced killer (CIK) cells, human cord blood CD34+cells, B cells, or cell lines such as JurkatT cell line.

The payload can include a small chemical molecule, a peptide or protein,or a nucleic acid. The small chemical molecule can be less than 1,000Da. The chemical molecule can include MitoTracker® Red CMXRos, propidiumiodide, methotrexate, and/or DAPI (4′,6-diamidino-2-phenylindole). Thepeptide can be about 5,000 Da. The peptide can include ecallantide undertrade name Kalbitor, is a 60 amino acid polypeptide for the treatment ofhereditary angioedema and in prevention of blood loss in cardiothoracicsurgery), Liraglutide (marketed as the brand name Victoza, is used forthe treatment of type II diabetes, and Saxenda for the treatment ofobesity), and Icatibant (trade name Firazyer, a peptidomimetic for thetreatment of acute attacks of hereditary angioedema). Thesmall-interfering ribonucleic acid (siRNA) molecule can be about 20-25base pairs in length, or can be about 10,000-15,000 Da. The siRNAmolecule can reduces the expression of any gene product, e.g., knockdownof gene expression of clinically relevant target genes or of modelgenes, e.g., glyceraldehyde-3phosphate dehydrogenase (GAPDH) siRNA,GAPDH siRNA-FITC, cyclophilin B siRNA, and/or lamin siRNA. Proteintherapeutics can include peptides, enzymes, structural proteins,receptors, cellular proteins, or circulating proteins, or fragmentsthereof. The protein or polypeptide be about 100-500,000 Da, e.g.,1,000-150,000 Da. The protein can include any therapeutic, diagnostic,or research protein or peptide, e.g., beta-lactoglobulin, ovalbumin,bovine serum albumin (BSA), and/or horseradish peroxidase. In otherexamples, the protein can include a cancer-specific apoptotic protein,e.g., Tumor necrosis factor-related apoptosis inducing protein (TRAIL).

An antibody is generally be about 150,000 Da in molecular mass. Theantibody can include an anti-actin antibody, an anti-GAPDH antibody, ananti-Src antibody, an anti-Myc ab, and/or an anti-Raf antibody. Theantibody can include a green fluorescent protein (GFP) plasmid, a GLucplasmid and, and a BATEM plasmid. The DNA molecule can be greater than5,000,000 Da. In some examples, the antibody can be a murine-derivedmonoclonal antibody, e.g., ibritumomab tiuxetin, muromomab-CD3,tositumomab, a human antibody, or a humanized mouse (or other species oforigin) antibody. In other examples, the antibody can be a chimericmonoclonal antibody, e.g., abciximab, basiliximab, cetuximab,infliximab, or rituximab. In still other examples, the antibody can be ahumanized monoclonal antibody, e.g., alemtuzamab, bevacizumab,certolizumab pegol, daclizumab, gentuzumab ozogamicin, trastuzumab,tocilizumab, ipilimumamb, or panitumumab. The antibody can comprise anantibody fragment, e.g., abatecept, aflibercept, alefacept, oretanercept. The invention encompasses not only an intact monoclonalantibody, but also an immunologically-active antibody fragment, e.g., aFab or (Fab)2 fragment; an engineered single chain Fv molecule; or achimeric molecule, e.g., an antibody which contains the bindingspecificity of one antibody, e.g., of murine origin, and the remainingportions of another antibody, e.g., of human origin.

The payload can include a therapeutic agent. A therapeutic agent, e.g.,a drug, or an active agent”, can mean any compound useful fortherapeutic or diagnostic purposes, the term can be understood to meanany compound that is administered to a patient for the treatment of acondition. Accordingly, a therapeutic agent can include, proteins,peptides, antibodies, antibody fragments, and small molecules.Therapeutic agents described in U.S. Pat. No. 7,667,004 (incorporatedherein by reference) can be used in the methods described herein. Thetherapeutic agent can include at least one of cisplatin, aspirin,statins (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin,rosuvastatin, simvastatin, promazine HCl, chloropromazine HCl,thioridazine HCl, Polymyxin B sulfate, chloroxine, benfluorex HCl andphenazopyridine HCl), and fluoxetine. The payload can include adiagnostic agent. The diagnostic agent can include a detectable label ormarker such as at least one of methylene blue, patent blue V, andindocyanine green. The payload can include a fluorescent molecule. Thepayload can include a detectable nanoparticle. The nanoparticle caninclude a quantum dot.

The population of cells can be substantially confluent, such as greaterthan 75 percent confluent. Confluency of cells refers to cells incontact with one another on a surface. For example, it can be expressedas an estimated (or counted) percentage, e.g., 10% confluency means that10% of the surface, e.g., of a tissue culture vessel, is covered withcells, 100% means that it is entirely covered. For example, adherentcells grow two dimensionally on the surface of a tissue culture well,plate or flask. Non-adherent cells can be spun down, pulled down by avacuum, or tissue culture medium aspiration off the top of the cellpopulation, or removed by aspiration or vacuum removal from the bottomof the vessel. The population of cells can form a monolayer of cells.

The alcohol can be selected from methanol, ethanol, isopropyl alcohol,butanol and benzyl alcohol. The salt can be selected from NaCl, KCl,Na₂HPO₄, KH₂PO₄, and C₂H₃O₂NH. The sugar can include sucrose. Thebuffering agent can include4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid.

The present subject matter relates to a method for delivering moleculesacross a plasma membrane. The present subject matter finds utility inthe field of intra-cellular delivery, and has application in, forexample, delivery of molecular biological and pharmacologicaltherapeutic agents to a target site, such as a cell, tissue, or organ.The method of the present subject matter comprises introducing themolecule to an aqueous composition to form a matrix; atomizing thematrix into a spray; and contacting the matrix with a plasma membrane.

This present subject matter relates to a composition for use indelivering molecules across a plasma membrane. The present subjectmatter finds utility in the field of intra-cellular delivery, and hasapplication in, for example, delivery of molecular biological andpharmacological therapeutic agents to a target site, such as a cell,tissue, or organ. The composition of the present subject mattercomprises an alcohol; a salt; a sugar; a buffering agent; and ammoniumacetate.

In some implementations, demonstrated is a permeabilisation techniquethat facilitates intracellular delivery of molecules independent of themolecule and cell type. Nanoparticles, small molecules, nucleic acids,proteins and other molecules can be efficiently delivered intosuspension cells or adherent cells in situ, including primary cells andstem cells, with low cell toxicity and the technique is compatible withhigh throughput and automated cell-based assays.

The example methods described herein include a payload, wherein thepayload includes an alcohol. By the term “an alcohol” is meant apolyatomic organic compound including a hydroxyl (—OH) functional groupattached to at least one carbon atom. The alcohol may be a monohydricalcohol and may include at least one carbon atom, for example methanol.The alcohol may include at least two carbon atoms (e.g. ethanol). Inother aspects, the alcohol comprises at least three carbons (e.g.isopropyl alcohol). The alcohol may include at least four carbon atoms(e.g., butanol), or at least seven carbon atoms (e.g., benzyl alcohol).The example payload may include no more than 50% (v/v) of the alcohol,more preferably, the payload comprises 2-45% (v/v) of the alcohol, 5-40%of the alcohol, and 10-40% of the alcohol. The payload may include20-30% (v/v) of the alcohol.

Most preferably, the payload includes 25% (v/v) of the alcohol.Alternatively, the payload can include 2-8% (v/v) of the alcohol, or 2%of the alcohol. The alcohol may include ethanol and the payloadcomprises 5, 10, 20, 25, 30, or 40% (v/v) of the ethanol. Examplemethods may include methanol as the alcohol, and the payload may include5, 10, 20, 25, 30, or 40% (v/v) of the methanol. The payload may include2-45% (v/v) of methanol, 20-30% (v/v), or 25% (v/v) methanol.Preferably, the payload includes 20-30% (v/v) of methanol. Furtheralternatively, the alcohol is butanol and the payload comprises 2, 4, or8% (v/v) of the butanol.

In some aspects of the present subject matter, the payload is in ahypotonic solution or buffer. The payload solution may have an osmoticconcentration of 171 mOsm/L. According to example methods, the payloadsolution has an osmotic concentration of 171 mOsm/L at room temperature.

According to the present subject matter, the payload may include atleast one salt. The salt may be selected from NaCl, KCl, Na₂HPO₄,C₂H₃O₂NH₄ and KH₂PO₄. According to example methods, the payload includeseach of NaCl, KCl, Na₂HPO₄, and KH₂PO₄. The payload may include lessthan 46 mM salt. Further, the payload includes 2-35 mM salt, or 10-15 mMsalt (e.g., 12 mM salt). According to example methods, the salt is KCland the payload includes 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mMKCl, and more preferably 12 mM KCl.

According to example methods of the present subject matter, the payloadmay include a sugar (e.g., a sucrose, or a disaccharide). According toexample methods, the payload comprises less than 121 mM sugar, 6-91 mM,or 26-39 mM sugar. Still further, the payload includes 32 mM sugar(e.g., sucrose). Optionally, the sugar is sucrose and the payloadcomprises 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8, or 89.6 mM sucrose.

According to example methods of the present subject matter, the payloadmay include a buffering agent (e.g. a weak acid or a weak base). Thebuffering agent may include a zwitterion. According to example methods,the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid. The payload may comprise less than 19 mM buffering agent (e.g.,1-15 mM, or 4-6 mM or 5 mM buffering agent). According to examplemethods, the buffering agent is4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and the payloadcomprises 1, 2, 3, 4, 5, 10, 12, 14 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Further preferably,the payload comprises 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid.

According to example methods of the present subject matter, the payloadincludes ammonium acetate. The payload may include less than 46 mMammonium acetate (e.g., between 2-35 mM, 10-15 mM, ore 12 mM ammoniumacetate). The payload may include 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or33.6 mM ammonium acetate.

The methods described herein include a second aspect of the presentsubject matter, where a second payload (e.g. an aqueous solution)including 68 mM NaCl, 1.4 mM KCl, 5 mM Na₂HPO₄, and 0.9 mM KH₂PO₄ isprovided. The pH of the second payload may be pH 7.4.

The volume of aqueous solution performed by gas propelling the aqueoussolution may include compressed air (e.g. ambient air), otherimplementations may include inert gases, for example, helium, neon, andargon.

In certain aspects of the present subject matter, the population ofcells may include adherent cells (e.g., lung, kidney, immune cells suchas macrophages) or non-adherent cells (e.g., suspension cells).

In certain aspects of the present subject matter, the population ofcells may be substantially confluent, and substantially may includegreater than 75 percent confluent. In preferred implementations, thepopulation of cells may form a single monolayer.

According to example methods, the payload to be delivered has an averagemolecular weight of up to 20,000,000 Da. In some examples, the payloadto be delivered can have an average molecular weight of up to 2,000,000Da. In some implementations, the payload to be delivered may have anaverage molecular weight of up to 150,000 Da. In furtherimplementations, the payload to be delivered has an average molecularweight of up to 15,000 Da, 5,000 Da or 1,000 Da.

The payload to be delivered across the plasma membrane of a cell mayinclude a small chemical molecule, a peptide or protein, apolysaccharide or a nucleic acid or a nanoparticle. A small chemicalmolecule may be less than 1,000 Da, peptides may have molecular weightsabout 5,000 Da, siRNA may have molecular weights around 15,000 Da,antibodies may have molecular weights of about 150,000 Da and DNA mayhave molecular weights of greater than or equal to 5,000,000 Da.

According to example methods, the payload includes 3.0-150.0 μM of amolecule to be delivered, more preferably, 6.6-150.0 μM molecule to bedelivered (e.g. 3.0, 3.3, 6.6, or 150.0 μM molecule to be delivered). Insome implementations, the payload to be delivered has an averagemolecular weight of up to 15,000 Da, and the payload includes 3.3 μMmolecules to be delivered.

According to example methods, the payload to be delivered has an averagemolecular weight of up to 15,000 Da, and the payload includes 6.6 μM tobe delivered. In some implementations, the payload to be delivered hasan average molecular weight of up to 1,000 Da, and the payload includes150.0 μM to be delivered.

Aspects of the present subject matter provide for the payload to bedelivered to have an average molecular weight of up to 15,000 Da. Thepayload may include an aqueous solution having an osmotic concentrationof 171 mOsm/L at room temperature and a pH of about 7.4; and including25% (v/v) of ethanol; 12 mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 12 mM ammoniumacetate; and 6.6 μM molecules to be delivered.

According to example methods, the payload to be delivered has an averagemolecular weight of up to 15,000 Da. The payload may include an aqueoussolution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and includes 20% (v/v) of methanol;12 mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 12 mM ammoniumacetate; and 6.6 μM molecules to be delivered.

In some implementations, the molecule to be delivered has an averagemolecular weight of up to 15,000 Da. The payload may include an aqueoussolution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and includes 25% (v/v) of methanol;12 mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 12 mM ammoniumacetate; and 6.6 μM molecules to be delivered.

According to example methods, the payload to be delivered has an averagemolecular weight of up to 1,000 Da, the payload includes an aqueoussolution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and includes 25% (v/v) of ethanol; 12mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 12 mM ammoniumacetate; and 150 μM molecules to be delivered.

In some implementations, the molecule to be delivered has an averagemolecular weight of up to 1,000 Da. According to example methods, thepayload may include an aqueous solution having an osmotic concentrationof 171 mOsm/L at room temperature and a pH of about 7.4; and includes25% (v/v) of ethanol; 34 mM NaCl. 0.7 mM KCl, 2.5 mM Na₂HPO₄, and 0.5 mMKH₂PO₄; and 150.0 μM molecules to be delivered.

The payload to be delivered can have an average molecular weight of upto 1,000 Da. According to example methods, the payload can include anaqueous solution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and can include 2% (v/v) of butanol;12 mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 12 mM ammoniumacetate; and 150 μM molecules to be delivered.

According to further aspects of the present subject matter, a method fordelivering molecules of more than one molecular weight across a plasmamembrane is provided; the method including the steps of: introducing themolecules of more than one molecular weight to an aqueous solution; andcontacting the aqueous solution with a plasma membrane.

In some implementations, the method includes introducing a firstmolecule having a first molecular weight and a second molecule having asecond molecular weight to the payload, wherein the first and secondmolecules may have different molecular weights, or wherein, the firstand second molecules may have the same molecular weights. According toexample methods, the first and second molecules may be differentmolecules.

In some implementations, the payload to be delivered may include atherapeutic agent, or a diagnostic agent, including, for example,cisplatin, aspirin, various statins (e.g., pitavastatin, atorvastatin,lovastatin, pravastatin, rosuvastatin, simvastatin, promazine HCl,chloropromazine HCl, thioridazine HCl, Polymyxin B sulfate, chloroxine,benfluorex HCl and phenazopyridine HCl), and fluoxetine. Othertherapeutic agents include antimicrobials (aminoclyclosides (e.g.gentamicin, neomycin, streptomycin), penicillins (e.g., amoxicillin,ampicillin), glycopeptides (e.g., avoparcin, vancomycin), macrolides(e.g., erythromycin, tilmicosin, tylosin), quinolones (e.g.,sarafloxacin, enrofloxin), streptogramins (e.g., viginiamycin,quinupristin-dalfoprisitin), carbapenems, lipopeptides, oxazolidinones,cycloserine, ethambutol, ethionamide, isoniazrid, para-aminosalicyclicacid, and pyrazinamide). In some examples, an anti-viral (e.g.,Abacavir, Aciclovir, Enfuvirtide, Entecavir, Nelfinavir, Nevirapine,Nexavir, Oseltamivir Raltegravir, Ritonavir, Stavudine, andValaciclovir). The therapeutic may include a protein-based therapy forthe treatment of various diseases, e.g., cancer, infectious diseases,hemophilia, anemia, multiple sclerosis, and hepatitis B or C.

Additional exemplary payloads can also include detectable markers orlabels such as methylene blue, Patent blue V, and Indocyanine green.

The methods described herein may also include the payload including of adetectable moiety, or a detectable nanoparticle (e.g., a quantum dot).The detectable moiety may include a fluorescent molecule or aradioactive agent (e.g., ¹²⁵I) When the fluorescent molecule is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, p-phthaldehyde and fluorescamine. Themolecule can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the molecule using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA). The molecule also can be detectably labeled by coupling itto a chemiluminescent compound. The presence of thechemiluminescent-tagged molecule is then determined by detecting thepresence of luminescence that arises during the course of chemicalreaction. Examples of particularly useful chemiluminescent labelingcompounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

In one aspect, the present subject matter describes cells attached to asolid support, (e.g., a strip, a polymer, a bead, or a nanoparticle).The support or scaffold may be a porous or non-porous solid support.Well-known supports or carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, gabbros, and magnetite. The natureof the carrier can be either soluble to some extent or insoluble for thepurposes of the present subject matter. The support material may havevirtually any possible structural configuration. Thus, the supportconfiguration may be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, or test strip,etc. Preferred supports include polystyrene beads.

In other aspects, the solid support comprises a polymer, to which cellsare chemically bound, immobilized, dispersed, or associated. A polymersupport may be a network of polymers, and may be prepared in bead form(e.g., by suspension polymerization). The cells on such a scaffold canbe sprayed with payload containing aqueous solution according to theinvention to deliver desired compounds to the cytoplasm of the scaffold.Exemplary scaffolds include stents and other implantable medical devicesor structures.

The present subject matter further relates to apparatus, systems,techniques and articles for delivery of payloads across a plasmamembrane. The present subject matter also relates to an apparatus fordelivering payloads such as proteins or protein complexes across aplasma membrane. The current subject matter may find utility in thefield of intra-cellular delivery, and has application in, for example,delivery of molecular biological and pharmacological therapeutic agentsto a target site, such as a cell, tissue, or organ.

In some implementations, an apparatus for delivering a payload across aplasma membrane can include an atomizer having at least one atomizeremitter and a support oriented relative to the atomizer. The methodfurther comprises the step of atomizing the payload prior to contactingthe plasma membrane with the payload.

The atomizer can be selected from a mechanical atomizer, an ultrasonicatomizer, an electrospray, a nebuliser, and a Venturi tube. The atomizercan be a commercially available atomizer. The atomizer can be anintranasal mucosal atomization device. The atomizer can be an intranasalmucosal atomization device commercially available from LMA Teleflex ofNC, USA. The atomizer can be an intranasal mucosal atomization devicecommercially available from LMA Teleflex of NC, USA under cataloguenumber MAD300.

The atomizer can be adapted to provide a colloid suspension of particleshaving a diameter of 30-100 μm prior to contacting the plasma membranewith the payload. The atomizer can be adapted to provide a colloidsuspension of particles having a diameter of 30-80 μm. The atomizer canbe adapted to provide a colloid suspension of particles having adiameter of 50-80 μm.

The atomizer can include a gas reservoir. The atomizer can include a gasreservoir with the gas maintained under pressure. The gas can beselected from air, carbon dioxide, and helium. The gas reservoir caninclude a fixed pressure head generator. The gas reservoir can be influid communication with the atomizer emitter. The gas reservoir caninclude a gas guide, which can be in fluid communication with theatomizer emitter. The gas guide can be adapted to allow the passage ofgas therethrough. The gas guide can include a hollow body. The gas guidecan be a hollow body having open ends. The gas guide can include ahollow body having first and second open ends. The gas guide can be ahollow body having first and second opposing open ends. The diameter ofthe first open end can be different to the diameter of the second openend. The diameter of the first open end can be different to the diameterof the second open end. The diameter of the first open end can begreater than the diameter of the second open end. The first open end canbe in fluid communication with the gas reservoir. The second open endcan be in fluid communication with the atomizer emitter.

The apparatus can include a sample reservoir. The sample reservoir canbe in fluid communication with the atomizer. The sample reservoir can bein fluid communication with the atomizer emitter. The gas reservoir andthe sample reservoir can both be in fluid communication with theatomizer emitter.

The apparatus can include a sample valve located between the samplereservoir and the gas reservoir. The apparatus can include a samplevalve located between the sample reservoir and the gas guide. The samplevalve can be adapted to adjust the sample flow from the samplereservoir. The sample valve can be adapted to allow continuous orsemi-continuous sample flow. The sample valve can be adapted to allowsemi-continuous sample flow. The sample valve can be adapted to allowsemi-continuous sample flow of a defined amount. The sample valve isadapted to allow semi-continuous sample flow of 0.5-100 μL. The samplevalve can be adapted to allow semi-continuous sample flow of 10 μL. Thesample valve can be adapted to allow semi-continuous sample flow of 1 μLto an area of 0.065-0.085 cm².

The atomizer and the support can be spaced apart. The support caninclude a solid support. The support can include a plate includingsample wells. The support can include a plate including sample wellsselected from 1, 6, 9, 12, 24, 48, 384, and 1536 wells. The solidsupport can be formed from an inert material. The solid support can beformed from a plastic material, or a metal or metal alloy, or acombination thereof. The support can include a heating element. Thesupport can include a resistive element. The support can be reciprocallymountable to the apparatus. The support can be reciprocally movablerelative to the apparatus. The support can be reciprocally movablerelative to the atomizer. The support can be reciprocally movablerelative to the atomizer emitter. The support can include a supportactuator to reciprocally move the support relative to the atomizer. Thesupport can include a support actuator to reciprocally move the supportrelative to the atomizer emitter. The support can include a supportactuator to reciprocally move the support relative to the longitudinalaxis of the atomizer emitter. The support can include a support actuatorto reciprocally move the support transverse to the longitudinal axis ofthe atomizer emitter.

The longitudinal axis of the spray zone can be coaxial with thelongitudinal axis or center point of the support and/or the circularwell of the support, to which the payload is to be delivered. Thelongitudinal axis of the atomizer emitter can be coaxial with thelongitudinal axis or center point of the support and/or the circularwell of the support. The longitudinal axis of the atomizer emitter, thelongitudinal axis of the support, and the longitudinal axis of the sprayzone can be each coaxial. The longitudinal length of the spray zone maybe greater than the diameter (may be greater than double) of thecircular base of the spray zone (e.g., the area of cells to which thepayload is to be delivered).

The apparatus can include a valve located between the gas reservoir andthe atomizer. The valve can be an electromagnetically operated valve.The valve can be a solenoid valve. The valve can be a pneumatic valve.The valve can be located at the gas guide. The valve can be adapted toadjust the gas flow within the gas guide. The valve can be adapted toallow continuous or semi-continuous gas flow. The valve can be adaptedto allow semi-continuous gas flow. The valve can be adapted to allowsemi-continuous gas flow of a defined time interval. The valve can beadapted to allow semi-continuous gas flow of a one second time interval.The apparatus can include at least one filter. The filter can include apore size of less than 10 μm. The filter can have a pore size of 10 μm.The filter can be located at the gas guide. The filter can be in fluidcommunication with the gas guide.

The apparatus can include at least one regulator. The regulator can bean electrical regulator. The regulator can be a mechanical regulator.The regulator can be located at the gas guide. The regulator can be influid communication with the gas guide. The regulator can be aregulating valve. The pressure within the gas guide can be 1.0-2.0 bar.The pressure within the gas guide can be 1.5 bar. The pressure withinthe gas guide can be 1.0-2.0 bar, and the distance between the atomizerand the support can be less than or equal to 31 mm. The pressure withinthe gas guide can be 1.5 bar, and the distance between the atomizer andthe support can be 31 mm. The pressure within the gas guide can be 0.05bar per millimeter distance between the atomizer and the support. Theregulating valve can be adapted to adjust the pressure within the gasguide to 1.0-2.0 bar. The regulating valve cam be adapted to adjust thepressure within the gas guide to 1.5 bar. The or each regulating valvecan be adapted to maintain the pressure within the gas guide at 1.0-2.0bar. The or each regulating valve can be adapted to maintain thepressure within the gas guide at 1.5 bar.

The apparatus can include two regulators. The apparatus can includefirst and second regulators. The first and second regulator can belocated at the gas guide. The first and second regulator can be in fluidcommunication with the gas guide. The first regulator can be locatedbetween the gas reservoir and the filter. The first regulator can beadapted to adjust the pressure from the gas reservoir within the gasguide to 2.0 bar. The first regulator can be adapted to maintain thepressure within the gas guide at 2.0 bar. The second regulator can belocated between the filter and the valve.

The atomizer emitter can be adapted to provide a conical spray zone(e.g., a generally circular conical spray zone). The atomizer emittercan be adapted to provide a 30° conical spray zone. The apparatusfurther can include a microprocessor to control any or all parts of theapparatus. The microprocessor can be arranged to control any or all ofthe sample valve, the support actuator, the valve, and the regulator.The apparatus can include an atomizer having at least one atomizeremitter; and a support oriented relative to the atomizer; the atomizercan be selected from a mechanical atomizer, an ultrasonic atomizer, anelectrospray, a nebuliser, and a Venturi tube. The atomizer can beadapted to provide a colloid suspension of particles having a diameterof 30-100 μm. The apparatus can include a sample reservoir and a gasguide, and a sample valve located between the sample reservoir and thegas guide. The sample valve can be adapted to allow semi-continuoussample flow of 10-100 μL. The atomizer and the support can be spacedapart and define a generally conical spray zone there between; and thedistance between the atomizer and the support can be approximatelydouble the diameter of the circular base of the area of cells to whichmolecules are to be delivered; the distance between the atomizer and thesupport can be 31 mm and the diameter of the circular base of the areaof cells to which molecules are to be delivered can be 15.5 mm. Theapparatus can include a gas guide and the pressure within the gas guideis 1.0-2.0 bar. The apparatus can include at least one filter having apore size of less than 10 μm.

The details of one or more variations of the subject matter describedherein are set forth in the accompanying drawings and the descriptionbelow. Other features and advantages of the subject matter describedherein will be apparent from the description and drawings, and from theclaims.

DESCRIPTION OF DRAWINGS

Non-limiting embodiments of the present subject matter will now bedescribed with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of an apparatus for implementing a methodaccording to the present subject matter.

FIG. 2 is a perspective view of an apparatus for implementing a methodaccording to the present subject matter.

FIG. 3 is a process flow diagram illustrating a process of producingcolloidal droplets for delivering a sample to the cytoplasm of one ormore target cells.

FIG. 4 is a graph illustrating the effect of delivering a moleculehaving an average molecular weight of up to 15,000 Da across a plasmamembrane according to the present subject matter.

FIG. 5 is a graph illustrating the effect of delivering a moleculehaving an average molecular weight of up to 1,000 Da across a plasmamembrane according to the present subject matter.

FIG. 6A is a photomicrograph illustrating delivery of a molecule havingan average molecular weight of 668 Da.

FIG. 6B is a photomicrograph illustrating delivery of a molecule havingan average molecular weight of 40,000 Da.

FIG. 6C is a photomicrograph illustrating an overlay of FIGS. 6A and 6B;illustrating the delivery of a molecule having an average molecularweight of 668 Da (FIG. 6A) and 40,000 Da (FIG. 6B).

FIG. 7 is a graph illustrating the effect of delivering a moleculehaving an average molecular weight of up to 500,000 Da across a plasmamembrane according to the present subject matter.

FIG. 8 is a graph illustrating the effect of contacting cells with asecond composition including 68 mM NaCl, 1.4 mM KCl, 5 mM Na₂HPO₄, and0.9 mM KH₂PO₄ according to the present subject matter.

FIG. 9 is a photomicrograph illustrating delivering molecules of varyingmolecular weights using a method according to the present subjectmatter.

FIG. 10 is a graph illustrating the effect of solute content on thedelivery of a molecule having an average molecular weight of up to15,000 Da.

FIG. 11 is a graph illustrating the effect of alcohol concentration onthe delivery of a molecule having an average molecular weight of up to15,000 Da.

FIG. 12 is a graph illustrating the effect of alcohol concentration onthe delivery of a molecule having an average molecular weight of up to15,000 Da.

FIG. 13 is a graph illustrating the effect of salt concentration on thedelivery of a molecule having an average molecular weight of up to15,000 Da.

FIG. 14 is a graph illustrating the effect of alcohol concentration onthe delivery of a molecule having an average molecular weight of up to1,000 Da.

FIG. 15 is a graph illustrating the effect of alcohol concentration onthe delivery of a molecule having an average molecular weight of up to1,000 Da.

FIG. 16 is a graph illustrating the effect of alcohol concentration onthe delivery of a molecule having an average molecular weight of up to1,000 Da.

FIG. 17 is a photomicrograph illustrating the micropipette-mediateddelivery of payloads in 200 μL of delivery solution to A549 cells in24-well plates, and viewed by fluorescent microscopy. Propidium iodide(PI) uptake was visible throughout the cell population, but no uptake ofsiRNA-FITC or 10 kDa Dextran Alexa488 was apparent. All photomicrographsare shown at a 10× magnification.

FIG. 18 is a photomicrograph illustrating the micropipette-mediateddelivery of payloads in 20 μL, of delivery solution to A549 cells in24-well plates, and viewed by fluorescent microscopy. Uptake of PI wasapparent in some areas of the well but not in others. Allphotomicrographs are shown at a 10× magnification.

FIG. 19 is a graph illustrating Micropipette-mediated delivery ofpayloads in either 200 μl or 20 μl delivery solution to A549 cells in24-well plates. Uptake efficiency was measured by flow cytometry (5% mbar) and toxicity was measured by lactate dehydrogenase (LDH) releasecompared to lysed cells positive control. When payloads were deliveredin 200 μl, uptake was detected by flow cytometry but toxicity levelswere high (40-50%). When payloads were delivered in 20 μl, toxicity wasreduced but uptake was also reduced and was inconsistent; (n=3).MP=micropipette; PI=propidiumiodide; Dex=10 kDadextran-Alexa488;SBO=spray buffer only.

FIG. 20 is an image showing the instrument. An instrument wasconstructed that would enable spray mediated delivery of the deliverysolution to cells. The instrument comprised an air compressor thatdelivered compressed air to a sprayhead which was held in position on aretort stand. The culture plate was positioned on a stage below thesprayhead.

FIG. 21 is a photomicrograph illustrating delivery of 10 kDadextran-Alexa488 to A549 cells via an example implementation of thepresent subject matter. 10 kDa dextran-Alexa488 was successfullydelivered to A549 cells using the method of the current subject matter.Uptake was evident across the cell monolayer. A 10× magnification isshown in the photomicrograph.

FIG. 22 is a graph showing the efficiency and toxicity of the deliverymethod of the current subject matter. Efficiency levels of greater than50% delivery of 10-kDa dextran-Alexa488 were achieved in A549 cells.Toxicity levels were similar to untreated controls. When the deliverysolution containing payload was spiked into the culture medium, somepositive cells were also detected (n=3).

FIG. 23 is a photomicrograph showing the time course of permeabilisationin A549 cells. Delivery solution was sprayed in the absence of payload,and PI was subsequently added to the culture medium at time points up to60 minutes post-spray to detect permeabilised cells. While PI uptake wasvisible at 5 min post-spray, the number of PI-positive cells wassubstantially reduced by 30 minutes and 60 minutes post-spray. A 10×magnification is shown in the photomicrograph.

FIG. 24 is a graph illustrating the effect of the distance between thesprayhead and the cells on delivery efficiency and cell toxicity.10-kDadextran-Alexa488 was delivered to A549 cells. The distance betweenthe sprayhead and the cells was varied (including 21 mm, 31 mm and 41mm). A distance of 31 mm was optimal for both efficiency and toxicity(n=3).

FIG. 25 is a graph illustrating the effect of spray pressure on deliveryefficiency and cell toxicity. 10-kDadextran-Alexa488 was delivered toA549 cells, and the spray pressure was varied (including 0.5 bar, 1.5bar and 2.5 bar). A pressure of 1.5 bar was optimal for both deliveryefficiency and cell toxicity (n=3).

FIG. 26 is a graph illustrating the effect of the volume of deliverysolution on delivery efficiency and cell toxicity.10-kDadextran-Alexa488 was delivered to A549 cells at 80-90% confluencyin 48-wellplates, and the volume of delivery solution was varied(including 5 μL, 10 μL and 20 μL). A volume of 10 μl was optimal forboth delivery efficiency and cell toxicity (n=3).

FIG. 27 is a graph illustrating the effect of the volume of deliverysolution on delivery efficiency and cell toxicity.10-kDadextran-Alexa488 was delivered to CHO cells at 80-90% confluencyin 48-wellplates, and the volume of delivery solution was varied(including 5 μL, 10 μL and 20 μL). A volume of 10 μl was optimal forboth delivery efficiency and cell toxicity (n=3).

FIG. 28 is a graph illustrating the effect of ethanol concentration ondelivery efficiency and cell toxicity. 10-kDadextran-Alexa488 wasdelivered to A549 cells, and the concentration of ethanol in thedelivery solution was varied. A concentration of 25% was optimal forboth efficiency and toxicity (n=3).

FIG. 29 is a photomicrograph illustrating the delivery of a wide rangeof molecular sizes of dextrans, including very high molecular weightdextran (2,000 kDa) that can be delivered by the method of the currentsubject matter. The photomicrograph shows a 10× magnification.

FIG. 30 is a photomicrograph illustrating the delivery of a wide rangeof molecular sizes of molecules, including full length antibodies thatcan be delivered by methods of the current subject matter. Thephotomicrograph shows a 10× magnification.

FIG. 31 is a photomicrograph illustrating the effect of co-delivery of4′,6-diamidino-2-phenylindole (DAPI), Mitotracker Red CMXRos andPhalloidin-Alexa488 to A549 cells.

FIG. 32 is a photomicrograph illustrating the effect of co-delivery ofboth 10 kDa dextran-Alexa488 and DAPI to A549 cells. The photomicrographshows a 10× magnification.

FIG. 33 is a photomicrograph illustrating the effect of delivery of GFPmRNA that was sprayfected into A549 cells. GFP protein expression wasobserved by fluorescence microscopy. The photomicrograph shows a 10×magnification.

FIG. 34 is a bar graph showing the quantification (by luminometry) ofthe expression of luciferase when luciferase mRNA was delivered intoA549 cells using exemplary methods of the current subject matter.

FIG. 35 is a photomicrograph illustrating the effect of delivery ofdelivery of pGFP plasmid DNA that was sprayfected into A549 cells. Theexpression of GFP protein was observed by fluorescence microcopy. Thephotomicrograph shows a 10× magnification.

FIG. 36 is a bar graph showing the quantification (by luminometry) ofthe expression of luciferase when pGluc plasmid DNA was delivered intoA549 cells using exemplary methods of the current subject matter.

FIG. 37 is a photomicrograph illustrating the effect when 10 kDadextran-Alexa488 was delivered into primary fibroblasts. Dextran wasvisible in fibroblasts by fluorescence microscopy. A 10× magnificationis shown.

FIG. 38 is a bar graph showing the effect of efficiency and toxicity ofdelivery of 10 kDa dextran Alexa488 delivered into primary fibroblasts,as quantified by flow cytometry and an LDH assay, respectively.

FIG. 39 is a photomicrograph illustrating the effect when 10 kDadextran-Alexa488 was delivered into mesenchymal stem cells (MSC).Dextran was visible in MSCs by fluorescence microscopy, at 10×magnification.

FIG. 40 is a bar graph showing the effect of efficiency and toxicity ofdelivery of 10 kDa dextran Alexa488 delivered into MSCs, as quantifiedby flow cytometry and an LDH assay, respectively.

FIG. 41A is a photomicrograph illustrating the effect of delivery ofsiRNA (top panel), BSA (middle panel), and OVA (bottom panel) into U226human multiple myeloma cells.

FIG. 41B is a photomicrograph illustrating the effect of delivery ofDAPI (top panel) and MItotracker Red (middle panel) to Jurkat cells.Additionally, mRNA for green fluorescent protein (GFP) was delivered toJurkat cells, and GFP expression was observed at 24 hours post-delivery.

FIG. 42 is a bar graph illustrating the effect of delivery efficiencyand cell toxicity of siRNA (top panel), BSA (middle panel), and OVA(bottom panel) into U226 human multiple myeloma cells, as quantified byflow cytometry and an LDH assay, respectively.

FIG. 43 is a photomicrograph showing the effects of delivery of proteinsinto Chinese hamster ovary (CHO) cells. A wide range of proteins werelabeled with FITC and delivered into CHO cells, includingβ-lactoglobulin, horseradish peroxidase (HRP), ovalbumin, bovine serumalbumin (BSA), catalase, and apoferritin.

FIG. 44 is a graph showing the efficiencies of delivery of a wide rangeof proteins into CHO cells by the exemplary methods of the currentsubject matter. The Efficiencies were quantified by flow cytometry.

FIG. 45 is a photomicrograph showing the immunofluorescence detection ofovalbumin-FITC delivered into CHO cells. Delivery of ovalbumin-FITC intoCHO cells was validated by immunofluorescence using an anti-ovalbuminantibody.

FIG. 46 is a graph showing the dose response for delivery ofbeta-lactoglobulin into CHO cells. Increasing efficiency of delivery wasseen with increasing concentrations of beta-lactoglobulin-FITC deliveredto CHO cells by methods of the current subject matter (n=3).

FIG. 47 is a photomicrograph illustrating the activity and localizationof HRP delivered to cells. Alexa488-labeled tyramide substrate was usedto demonstrate activity and localization of HRP in CHO cells followingdelivery of HRP by methods of the current subject matter.

FIG. 48 is a bar graph illustrating the increased production offluorescent DCF product detected with increased dose of HRP deliveredinto CHO cells.

FIG. 49 is a bar graph indicating that the LDH analysis demonstratedthat the assay was not toxic to cells.

FIG. 50 is a photomicrograph illustrating the labeling of MSC withQ-dots for tracking studies. Primary MSCs were delivered with Q-dot 625in vitro.

FIG. 51 is a photomicrograph illustrating the labeling of MSC withQ-dots for tracking studies. MSCs were injected into mouse spleens exvivo. Q-dot fluorescence was analyzed using the Cryovis instrument.

FIG. 52 is a table illustrating approximate volume delivered per cellaccording to an example implementation of the currents subject matter.

FIG. 53 is a table illustrating approximate volume delivered per squaremicrometer of exposed cell surface area.

FIG. 54 is a table illustrating approximate average properties of somecell types.

FIG. 55 is a table showing experimentally calculated and measured areasof three different cell lines (A549, CHO, and MCSs).

FIG. 56A is a cross-sectional view of an illustration of a well having avolume of aqueous solution applied using a pipette.

FIG. 56B is a cross-sectional view of an illustrations of a well havinga volume of aqueous solution applied via a spray technique.

Like reference symbols in the various drawings indicate like elements

DETAILED DESCRIPTION

The present subject matter provides for vector-free (e.g., viralvector-free) delivery of a payload across a plasma membrane. Inparticular, it has been discovered that intracellular delivery ofmaterials can be achieved by contacting a cell (and/or population ofcells) with an aqueous solution that includes an alcohol and thedelivery materials (e.g., the payload). The alcohol acts to permeabilisethe membrane to allow the payload to translocate across the membrane.But permanent or severe (e.g., irreversible) damage to the cell mayoccur (adversely affecting cell viability) when the volume of aqueoussolution that contacts the cell is too large and/or exposure occurs fortoo long a time. Conversely, intracellular delivery of materials is notachieved when the volume of aqueous solution that contacts the cell istoo small and/or exposure occurs for too short a time. Thus, to achievedelivery of a payload across a plasma membrane while maintaining cellviability, an appropriate volume of aqueous solution can be appliedand/or the length of exposure can be controlled.

The appropriate volume of aqueous solution that is contacted to apopulation of cells can vary based on the intended application, forexample, based on (e.g., be a function of) number of cells in thepopulation, exposed cell surface area, cell size, makeup of the aqueoussolution, payload, technique of contacting the aqueous solution to thepopulation of cells, and the like. In some implementations, the volumeof aqueous solution can be between 6.0×10⁻⁷ and 7.4×10⁻⁴ microliters percell (additional ranges are described elsewhere herein). These rangescorrespond to delivering between 0.5 microliters and 100 microliters ofaqueous solution to a well in a 48 well plate having a population ofcells arranged substantially in a monolayer (the cells having an averagediameter of 30 micrometers and 15 micrometers, respectfully). The volumeof aqueous solution can be between 2.6×10⁻⁹ and 1.1×10⁻⁶ microliter persquare micrometer of exposed surface area of the population of cells(additional ranges are described elsewhere herein). These rangescorrespond to delivering between 0.5 microliters and 100 microliters ofaqueous solution to a well in a 24 well plate and a 48 well plate,respectfully, and having a population of cells arranged substantially ina monolayer.

The technique for contacting the population of cells with the aqueoussolution can vary. For example, the aqueous solution can be pipettedonto the population of cells (for example, when the cells are arrangedin a well). For example, FIG. 56A is a cross-sectional view illustratinga well with a monolayer of cells having a volume of aqueous solutionapplied using a micropipette. When the aqueous solution is applied inthis manner, it may be unevenly distributed over the area of the welland, as a result, cells located near the center of the well (the regionindicated at 5605) are killed, while cells located near the outer edgesof the well (the region indicated at 5615) remain viable but exhibit noupdate of the payload. Cells in a region 5610 between the inner andouter regions (5605 and 5615, respectively) remain viable whileefficiently and reliably exhibiting uptake of the payload.

In some implementations, aqueous solution is sprayed onto the populationof cells. For example, FIG. 56B is a cross-sectional view illustrating awell having a volume of aqueous solution applied via a spray technique.The spray can evenly distribute the aqueous solution over the area ofthe well (region indicated at 5620). Cells treated in this manner (andas further described in detail herein) remain viable while efficientlyand reliably exhibiting uptake of payload across the cell membrane andinto the cytoplasm of the cell. Spraying can provide a means forcontacting a population of cells with the aqueous solution in acontrolled manner and has been shown to improve efficiency of deliveryof the payload and improve cell viability. The spray can be controlledto create discrete units (e.g., droplets) of volume that vary in size.For example, in an implementation, the discrete units of volume rangefrom 30-100 μm in diameter. Other sizes are possible and some variationsare described elsewhere herein.

Contacting of the population of cells with the aqueous solution(payload-containing) can be transient. In other words, the length oftime that the aqueous solution contacts the population of cells canvary. For example, the length of time of exposure can be at least 6seconds, 12 seconds, 30 seconds, and the like. Other lengths of time arepossible and some variations are described elsewhere herein. Becauseover exposure of cells to the aqueous solution can lead to lower cellviability, the population of cells can be washed with a buffer orculture medium after being exposed to the aqueous solution. The buffercan include or not include the payload. The buffer may be alcohol free.The cells can be washed with the buffer or culture medium to submerse orsuspend the population of cells. In some implementations, a gas may beblown across the cells to push the aqueous solution out of contact withthe cells, although over exposure of cells to gas may dehydrate thecells and lead to lower cell viability.

The aqueous solution can include H₂O, an alcohol, and the payload. Thealcohol can include methanol, ethanol, isopropyl alcohol, butanol orbenzyl alcohol. The aqueous solution can also include one or more of asugar, a salt and a buffering agent. The salt can be selected from NaCl,KCl, Na₂HPO₄ and KH₂PO₄. The sugar may include a disacharide, (e.g.,sucrose). The buffering agent may include a weak acid or a weak base andbe a zwitterion (e.g., (4-(2-hydroxyethyl) piperazineethanesulfonic acid(hepes)). The aqueous solution also can include ammonium acetate. Forexample, the aqueous solution is a hypotonic buffer, e.g., as describedby Medepalli et al., (Medepalli, K., et al., Nanotechnology 2013; 24:20,incorporated herein by reference in its entirety), 130 mM sucrose, 50 mMpotassium chloride, 50 mM potassium acetate, 20 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (hepes), pH 7.4. Insome examples, the buffer is modified to replace the potassium acetatewith ammonium acetate. In some examples, the buffer used for payloaddelivery does not include saponin.

Components of the aqueous solution can serve to disrupt the plasmamembrane of cells and allow for introduction of larger biologicalmolecules across the plasma membrane. For example, alcohols dissolvelipids within the plasma membrane, detergents create pores within theplasma membrane, and enzymes digest proteins to create pores within theplasma membrane.

Payload can be delivered into the cytoplasm of the cell, as well as tospecific cellular organelles (e.g., the nucleus and mitochondria). Thepayload can include any molecule suitable for and/or intended fordelivery. Molecules targeting the mitochondria are beneficial in anumber of diseases such as cancer and delivery of such molecules can berelated to functions of mitochondria including energy production andapoptosis. For example, fluorescently labeled (e.g., tagged) moleculescan be used to visualize the presence and location of mitochondrialcomponents, molecules that target mitochondrial permeability transition(MPT) (e.g., chemical inhibitors or peptides that deplete endogenousinhibitors of permeability transition pore complex (PTPC) opening),small chemical molecules that trigger mitochondrial permeabilitytransition (MPT), ligands that modulate the adenine nucleotidetranslocase (ANT), compounds that induce the overproduction of reactiveoxygen species (ROS), molecules that reverse the hyperglycolytic stateof cancer cells, molecules that prime cancer cells to the induction ofcell death, and the like.

The payload can include but is not limited to small chemical molecules,peptides, polypeptides, nucleic acid molecules antibodies, and DNA (e.g.plasmid DNA). Exemplary small chemical molecules include dextrans ofincreasing sizes up to 2,000,000 Da, including 3 kDa dextran, 40 kDadextran, 70 kDa dextran, or 500 kDa dextran, propidium iodide,4′,6-diamidino-2-phenylindole (DAPI), phallotoxin, MitoTracker Red orany combination thereof (for example, MitoTracker Red can beco-delivered with phallotoxin, as can 10 kDa dextran-Alexa488 and DAPI),methotrexate. Exemplary peptides, polypeptides, and proteins orfragments thereof include proteins of increasing size up to 500 kDa,including β-lactoglobulin, horseradish peroxidase, ovalbumin, bovineserum albumin, catalase and apoferritin). Exemplary peptides can includeecallantide, liraglutide and icatibant. Exemplary nucleic acids mayrefer to polynucleotides such as deoxyribonucleic acid (DNA), and whereappropriate ribonucleic acid (RNA). The term also includes equivalents,analogs of either DNA or RNA made from nucleotide analogs, and asapplicable to the present subject matter, may be single (sense orantisense) and double-stranded polynucleotides. Further nucleic acidexamples can include, an siRNA molecule (e.g., a GAPDH siRNA-FITC), acyclophilin B siRNA, or a lamin siRNA molecule), a double strandednucleic acid molecule, for example a double stranded RNA molecule, asingle stranded nucleic acid molecule, or an isolated nucleic acidmolecule). Example DNA payloads of the current subject matter includeDNA samples greater than or equal to 5,000,000 Da (e.g., pGFP, pGLuc,and p BATEM). Exemplary antibodies of the present subject matter caninclude an anti-actin antibody, an anti-GAPDH (Glyceraldehyde3-phosphate dehydrogenase) antibody, an anti-Src (proto-oncogenetyrosine-protein kinase Src) antibody, an anti-Myc antibody or ananti-Raf antibody. The antibodies of the present invention can bepolyclonal antisera or monoclonal antibodies. The present subject mattercan encompass not only an intact monoclonal antibody, but also anantibody fragment, e. g., a Fab or (Fab)2 fragment; an engineered singlechain FV molecule; or a chimeric molecule, e.g., an antibody whichcontains the binding specificity of one antibody, e.g., of murineorigin, and the remaining portions of another antibody, e.g., of humanorigin. The antibody may be a humanized antibody, wherein the antibodyis from a non-human species, whose protein sequence has been modified toincrease their similarity to antibody variants produced naturally inhumans. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are referred to herein as “import”residues, which are typically taken from an “import” antibody domain,particularly a variable domain.

The population of cells may include adherent cells that grow to form aconfluent (e.g., 75% confluent) monolayer on the growth surface area ofthe culture plate. Adherent cells can refer to cells, cell lines, andcell systems, whether prokaryotic or eukaryotic. Examples of cells thatcan be grown as adherent cells are liver or liver-derived (e.g., primaryhepatocytes and liver epithelial cells), epithelial cells, endothelialcells, neuronal cells, mesenchymal cells, pancreatic cells, skeletalmuscle cells, cardiomyocytes, carcinoma-derived cells, bone marrowcells, islets of Langerhans, adrenal medulla cells, osteoblasts,osteoclasts, T-lymphocytes, neurons, glial cells, ganglion cells,retinal cells, lung cells (e.g., A549 cells), fibroblasts, humanumbilical vein cells (HUVEC), fibroblasts, ovary cells (e.g., Chinesehamster ovary cells), embryonic kidney cells (e.g., human embryonickidney cells), and myoblast cells. Stem cells can also be used (e.g.,primary mesenchymal stem cells, neuronal stem cells, induced pluripotentstem cells, hematopoietic stem cells, mouse embryonic stem cells, andhuman embryonic stem cells).

The population of cells can also include non-adherent (e.g., suspension)cells. Exemplary non-adherent cells include stem cells (for example,hematopoietic stem cells), progenitor cells (for example hematopoieticprogenitor cells), T cells, natural killer (NK) cells, cytokine-inducedkiller (CIK) cells, human cord blood CD34+ cells, B cells and Jurkatcells.

The population of cells, as described herein, have been describedaccording to size (e.g., small, medium, and large), based on theircalculated diameter, as determined by the American Type CultureCollection (ATCC), Celeromics Technologies and other molecular biologyreferences. As referred to herein and in a non-limiting manner, in someexamples, a small cell has a diameter up to 10 μm (e.g., splenocytes orsmall neurons), a medium cell has a diameter between 10 μm and 20 μm(e.g., A549 cells, CHO cells or MCF7 cells), and a large cell has adimeter greater than 20 μm (e.g., K562 cells, and MSCs). Generally, thecategories (ranges) are not meant to be limiting, and experimentalconditions can affect the measured diameter of the cell.

In some implementations, the population of cells can be located on athree dimensional scaffold, which can be sprayed (or payload can bedelivered to the cells using another technique). The three dimensionalscaffold may be for use in ex vivo or in vivo use. It also contemplatedthat other aspects of the current subject matter can apply ex vivo or invivo.

Determining Volume as Function of Exposed Surface Area and Number ofCells

As described more fully herein, efficient delivery of payloads to A549cells in a well of a 48 well plate was achieved by contacting 10 μL ofaqueous solution to the population of cells via a spray technique andincubating the cells after approximately 2 minutes with a buffersolution. However, delivery can be achieved by contacting between 0.5 μLand 100 μL aqueous solution to cells of varying types in a well of a 48well plate, for example, contacting 0.5 μL, 5 μL, 10 μL, 15 μL, or 100μL of aqueous solution to a population of cells. But delivery is notlimited to using a well in a 48 (or 24) well plate and instead thevolume of aqueous solution to be contacted with a population of cellscan be a function of exposed cell surface area and/or number of cells inthe population. For example, to determine the volume of aqueous solutionto deliver per cell, the following describes a non-limiting examplemethod of computing the volume delivered per cell and per micrometer ofexposed cell surface area.

Exemplary adherent cells have an average diameter of about 10-30 μm. Forexample, A549 cells have an average diameter of 15 μm (corresponding to0.015 cm). Thus the average area of A549 cells is about 1.8×10⁻⁶ cm².The area in a single well of a standard 48-well cell culture plateincludes a growth area of 0.95 cm². Thus the number of cells (e.g., A549cells with a diameter of 15 μM is approximately about 500,000-500,500cells (e.g., 537, 691 cells), assuming 100% confluence. As an example,10 μL of the aqueous solution is delivered per well, thus approximately1.9×10⁻⁵ μL of aqueous solution were delivered to each cell (e.g., A549cells). Accordingly, about 1.9×10⁻⁵ microliters per cell was contactedwith the population of cells. Ranges of aqueous volume delivered percell was determined using aqueous volumes (e.g., 0.5 μL, 5 μL, 10 μL, 15μL and 100 μL) and various cell sizes (e.g., approximately 30 μm (MSCs),approximately 15 μm (A549 cells), and approximately 10 μm (U266 cells).These and additional example values are shown in FIG. 52 and FIG. 53.

As an exemplary calculation of the volume delivered as a function ofexposed surface are of the population of cells, the growth area of thecell culture plate was utilized. The surface area of a single wellwithin a 24 well plate is 19000 μm² (and 9500 μm² in a 48 well plate,and 3200 μm² in a 96 well plate). The range of aqueous volume deliveredper well was determined using aqueous volumes including 0.5 μL, 5 μL, 10μL, 15 μL and 100 μL. Thus the volume of aqueous solution delivered(e.g., 10 μL per well) per square micrometer includes 5.3×10⁻¹ μL perwell in a 24 well plate, 1.1×10⁻³ μL per well in a 48 well plate and3.1×10⁻³ μL per well in a 96 well plate. These and additional examplevalues are shown in FIG. 52. Additional Tables illustrating propertiesof some example cells are shown in FIG. 53.

Aqueous Solution and Delivery

The aqueous solution (also referred to herein as the composition)includes an alcohol selected from methanol, ethanol, isopropyl alcohol,butanol, and benzyl alcohol. The composition can include no more than50% (v/v) of the alcohol. In certain embodiments, the alcohol is ethanoland the composition includes 5, 10, 20, 25, 30, or 40% (v/v) of theethanol. Alternatively, the alcohol is methanol and the compositionincludes 5, 10, 20, 25, 30, or 40% (v/v) of the methanol. Further, thealcohol can be butanol and the composition includes 2, 4, or 8% (v/v) ofthe butanol. In preferred embodiments, the composition is an aqueoussolution including the alcohol. The composition is preferably hypotonic,having an osmotic concentration of 171 mOsm/L at room temperature and apH of about 7.4, and including at least one salt is selected from NaCl,KCl, Na₂HPO₄, and KH₂PO₄. In preferred embodiments, the salt is KCl andthe composition includes 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mMKCl. The composition can include a sugar, which can be sucrose and thecomposition can include 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8, or 89.6 mMsucrose. In such preferred embodiments, the composition additionallyincludes a buffering agent, which can be selected from a weak acid or aweak base. In a preferred embodiment, the buffering agent is4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and the compositionincludes 1, 2, 3, 4, 5, 10, 12, 14 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Additionally, thecomposition can include ammonium acetate, for example, 2.4, 4.8, 7.2,9.6, 12, 24, 28.8, or 33.6 mM ammonium acetate.

The present method can be used to deliver molecules having an averagemolecular weight of up to 2,000,000 Da, such as an average molecularweight of up to 150,000 Da, an average molecular weight of up to 15,000Da, an average molecular weight of up to 5,000 Da, and/or an averagemolecular weight of up to 1,000 Da.

The introducing step of the method can include introducing 3.0-150.0 μMmolecules to be delivered, optionally 3.3-150.0 μM, further optionally6.6-150.0 μM molecules to be delivered. Optionally, the introducing stepof the method includes introducing 3.0, 3.3, 6.6, or 150.0 μM moleculesto be delivered. When the molecule to be delivered has an averagemolecular weight of up to 15,000 Da, the introducing step can includeintroducing 3.3 μM molecules to be delivered, alternatively 6.6 μMmolecules to be delivered. Alternatively, when the molecule to bedelivered has an average molecular weight of up to 1,000 Da, theintroducing step includes introducing 150 μM molecules to be delivered.The amount of molecule introduced in the introducing step can beselected.

In a certain embodiment, the molecule to be delivered has an averagemolecular weight of up to 15,000 Da; and the method includes introducing6.6 μM molecules to be delivered to a composition including an aqueoussolution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and including 25% (v/v) of ethanol;12 mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; and 12 mM ammoniumacetate.

In another embodiment, the molecule to be delivered has an averagemolecular weight of up to 15,000 Da, and the method includes introducing6.6 μM molecules to be delivered to a composition including an aqueoussolution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and includes 20% (v/v) of methanol;12 mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; and 12 mM ammoniumacetate.

When the molecule to be delivered has an average molecular weight of upto 1,000 Da, the method can include introducing 150 μM molecules to bedelivered to a composition including an aqueous solution having anosmotic concentration of 171 mOsm/L at room temperature and a pH ofabout 7.4; and includes 25% (v/v) of ethanol; 12 mM KCl; 32 mM sucrose;5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; and 12 mMammonium acetate.

In another embodiment, the method includes introducing 150 μM moleculesto be delivered when the molecule to be delivered has an averagemolecular weight of up to 1,000 Da to a composition including an aqueoussolution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and including 20% (v/v) of methanol;12 mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; and 12 mM ammoniumacetate.

When the molecule to be delivered has an average molecular weight of upto 1,000 Da, the method can include introducing 150 μM molecules to bedelivered to a composition including an aqueous solution having anosmotic concentration of 171 mOsm/L at room temperature and a pH ofabout 7.4; and including 2% (v/v) of butanol; 12 mM KCl; 32 mM sucrose;5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; and 12 mMammonium acetate.

In an embodiment, the molecule to be delivered has an average molecularweight of up to 1,000 Da and the method includes introducing 150 μMmolecules to be delivered to a composition that includes an aqueoussolution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and including 25% (v/v) of ethanol;34 mM NaCl. 0.7 mM KCl, 2.5 mM Na₂HPO₄, and 0.5 mM KH₂PO₄.

According to example methods, the molecule to be delivered has anaverage molecular weight of up to 1,000 Da. In some implementations, thecomposition includes an alcohol, and may include at least two carbonatoms, (e.g., ethanol). The composition may include 2-45% (v/v) of thealcohol, optionally 20-30% (v/v) of the alcohol (e.g., 25% (v/v) of thealcohol). Still further optionally, the composition includes 2-45% (v/v)of ethanol, 20-30% (v/v) of ethanol, and 25% (v/v) ethanol. Preferably,the composition includes 20-30% (v/v) of ethanol. The composition can bea solution (e.g., an aqueous solution). In some implementations, thecomposition has an osmotic concentration of 171 mOsm/L, optionally atroom temperature. Preferably, the composition has an osmoticconcentration of 171 mOsm/L at room temperature. In someimplementations, the composition includes at least one salt selectedfrom NaCl, KCl, Na₂HPO₄, and KH₂PO₄. The composition can include lessthan 46 mM, (e.g., between 2-35 mM salt 10-15 mM salt, ore 12 mM salt).Preferably, the composition includes 12 mM KCl. Optionally, thecomposition has a pH of about 7.4. In some implementations, thecomposition includes a sugar, optionally a disaccharide (e.g., sucrose).The composition can include less than 121 mM sugar (e.g., 6-91 mM sugar,26-39 mM sugar, or 32 mM sugar). Further preferably, the composition mayinclude 32 mM sucrose. In some implementations, the composition caninclude a buffering agent selected from a weak acid and a weak base.Optionally, the buffering agent is4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Optionally, thecomposition includes less than 19 mM buffering agent, e.g., 1-14 mMbuffering agent, 4-6 mM buffering agent, or 5 mM buffering agent.Further preferably, the composition can include 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. According to examplemethods, the composition includes less than 46 mM ammonium acetate,e.g., 2-35 mM ammonium acetate, 10-15 mM ammonium acetate, or 12 mMammonium acetate. Preferably, the composition includes 150.0 μMmolecules to be delivered.

In some implementations, the molecule to be delivered has an averagemolecular weight of up to 1,000 Da. In some examples, the compositionincludes an aqueous solution having an osmotic concentration of 171mOsm/L at room temperature and a pH of about 7.4; and includes 25% (v/v)of ethanol; 12 mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 12 mM ammoniumacetate; and 150.0 μM molecules to be delivered.

According to example methods, the molecule to be delivered has anaverage molecular weight of up to 1,000 Da. In some implementations, thecomposition can include an alcohol including at least one carbon atoms,(e.g., methanol). Preferably, the composition includes ethanol. Thecomposition can include 2-45% (v/v) of the alcohol, 20-30% (v/v) of thealcohol, or 25% (v/v) of the alcohol. In some implementations, thecomposition includes 2-45% (v/v) of methanol, 20-30% (v/v) of methanol,or optionally 20% (v/v) methanol. Preferably, the composition includes20-30% (v/v) of methanol. In some implementations, the composition is asolution (e.g., an aqueous solution). Optionally or additionally, thecomposition has an osmotic concentration of 171 mOsm/L, optionally atroom temperature. Preferably, the composition has an osmoticconcentration of 171 mOsm/L at room temperature. In someimplementations, the composition includes at least one salt selectedfrom NaCl, KCl, Na₂HPO₄, and KH₂PO₄. The composition can include lessthan 46 mM, 2-35 mM salt, 10-15 mM salt, or 12 mM salt (e.g., 12 mMKCl). The composition can have a pH of about 7.4. In someimplementations, the composition includes a sugar, a disaccharide, orsucrose. The composition can include less than 121 mM sugar, 6-91 mMsugar, 26-39 mM sugar, or 32 mM sugar (e.g., 32 mM sucrose). In someimplementations, the composition includes a buffering agent selectedfrom a weak acid and a weak base. Optionally, the buffering agent is4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The composition caninclude less than 19 mM buffering agent, 1-14 mM buffering agent, 4-6 mMbuffering agent, and 5 mM buffering agent. Further preferably, thecomposition includes 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid. In some implementations, the composition includes less than 46 mMammonium acetate, 2-35 mM ammonium acetate, 10-15 mM ammonium acetate,or 12 mM ammonium acetate. Preferably, the composition includes 150.0 μMmolecules to be delivered.

The molecule to be delivered can have an average molecular weight of upto 1,000 Da. In some implementations, the composition includes anaqueous solution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and includes 20% (v/v) of methanol;12 mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 12 mM ammoniumacetate; and 150.0 μM molecules to be delivered.

According to example methods, the molecule to be delivered has anaverage molecular weight of up to 1,000 Da. The composition includes analcohol including at least four carbon atoms (e.g., butanol). Stillfurther, the composition includes 2-8% (v/v) of the alcohol, or 2, 4, or8% (v/v) of the alcohol (e.g. preferably, the composition includes 2%(v/v) of butanol). In some implementations, the composition is asolution (e.g., an aqueous solution). In some implementations, thecomposition has an osmotic concentration of 171 mOsm/L, optionally atroom temperature. Preferably, the composition has an osmoticconcentration of 171 mOsm/L at room temperature. In someimplementations, the composition includes at least one salt selectedfrom NaCl, KCl, Na2HPO4, and KH2PO4. The composition can include lessthan 46 mM, e.g., 2-35 mM salt, 10-15 mM salt, or 12 mM salt.Preferably, the composition includes 12 mM KCl. The composition can havea pH of about 7.4. In some implementations, the composition includes asugar, optionally a disaccharide, optionally sucrose. Optionally, thecomposition includes less than 121 mM sugar, 6-91 mM sugar, 26-39 mMsugar, or 32 mM sugar (e.g., 32 mM sucrose). In some implementations,the composition includes a buffering agent selected from a weak acid anda weak base. The buffering agent may be4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The composition caninclude less than 19 mM buffering agent, 1-14 mM buffering agent, 4-6mM, and 5 mM buffering agent. Further preferably, the compositionincludes 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Insome implementations, the composition includes less than 46 mM ammoniumacetate, 2-35 mM ammonium acetate, 10-15 mM ammonium acetate, or 12 mMammonium acetate. Preferably, the composition includes 150.0 μMmolecules to be delivered.

The molecule to be delivered can have an average molecular weight of upto 1,000 Da. In some implementations, the composition includes anaqueous solution having an osmotic concentration of 171 mOsm/L at roomtemperature and a pH of about 7.4; and includes 2% (v/v) of butanol; 12mM KCl; 32 mM sucrose; 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 12 mM ammoniumacetate; and 150.0 μM molecules to be delivered.

According to example methods, the molecule to be delivered has anaverage molecular weight of up to 1,000 Da. The composition may includean alcohol including at least two carbon atoms (e.g., ethanol), In someimplementations, the composition includes 2-45% (v/v) of the alcohol,(e.g., 20-30% (v/v) or 25% (v/v) of the alcohol). Further, thecomposition can include 2-45% (v/v) of ethanol (e.g., 20-30% (v/v), or25% (v/v) ethanol. In some implementations, the composition is asolution (e.g. an aqueous solution) According to example methods, thecomposition may have an osmotic concentration of 171 mOsm/L, (e.g., atroom temperature). According to example methods, the compositionincludes at least one salt selected from NaCl, KCl, Na₂HPO₄, and KH₂PO₄.The composition can include 34 mM NaCl or 0.7 mM KCl. The compositioncan include 2.5 mM Na₂HPO₄. The composition includes 0.5 mM KH₂PO₄.Preferably, the composition includes at least one of 34 mM NaCl. 0.7 mMKCl, 2.5 mM Na₂HPO₄, and 0.5 mM KH₂PO₄. Preferably, the compositionincludes 34 mM NaCl. 0.7 mM KCl, 2.5 mM Na₂HPO₄, and 0.5 mM KH₂PO₄.Preferably, the composition includes 150.0 μM molecules to be delivered.

In preferred embodiments, the method includes the steps of introducingthe molecule with a composition to form a matrix; atomizing the matrix;and contacting the matrix with a plasma membrane by delivering 1 μL ofmatrix in the form of an aerosol to an area of 0.065-0.085 cm².

The method can include contacting the matrix with a plasma membraneincludes delivering 1 μL of matrix to an area of 0.065-0.085 cm²,optionally to an area of 0.065-0.085 cm² of cells. In certainembodiments, contacting the matrix with a plasma membrane includesdelivering 10-100 μL, of matrix, optionally delivering 20 μL of matrix.In a preferable embodiment, contacting the matrix with a plasma membraneincludes delivering the matrix in the form of an aerosol, wherein themethod further includes the step of atomizing the matrix prior tocontacting the matrix with a plasma membrane. The atomizing step can beachieved using an atomizer as described herein. The method preferablyincludes atomizing the matrix to provide a colloid suspension ofparticles having a diameter of 30-100 μm prior to contacting the matrixwith a plasma membrane.

In the method of the subject matter, the atomizing step includesproviding a generally (circular) conical spray zone, as is schematicallyillustrated in FIG. 1. In preferred embodiments, the atomizing stepprovides a generally conical spray zone wherein the longitudinal lengthof the spray zone is greater than the diameter of the circular base ofthe sprayzone. In particularly preferred embodiments, the atomizing stepincludes providing a generally conical spray zone wherein thelongitudinal length of the spray zone is approximately double thediameter of the circular base of the sprayzone. The circular base of thesprayzone generally equates to the circular base of the area of cells towhich molecules are to be delivered. Accordingly, in a certainembodiment, the atomizing step includes providing a generally conicalspray zone wherein the longitudinal length of the spray zone is lessthan or equal to 31 mm and the diameter of the circular base of the areaof cells to which molecules are to be delivered is 15.5 mm. Thecontacting step is preferably conducted at a center point of the area towhich the matrix is to be delivered, for example, wherein thelongitudinal axis of the spray zone is coaxial with the longitudinalaxis or center point of the circular base of the area of cells to whichmolecules are to be delivered.

The method can include the further step of exposing the cells to whichthe matrix is to be delivered prior to contacting the matrix with aplasma membrane. In certain embodiments, the exposing step includesremoving a substantial amount of the liquid surrounding the cells, forexample by aspiration. In additionally preferred embodiments, the methodincludes the steps of introducing the molecule with a composition toform a matrix; atomizing the matrix; exposing the cells to which thematrix is to be delivered; and contacting the matrix with a plasmamembrane by delivering 1 μL of matrix in the form of an aerosol to anarea of 0.065-0.085 cm².

The method can further include incubating the exposed cells, optionallywith a buffer solution, such as phosphate buffered saline. Accordingly,an embodiment of the present subject matter defines a method includingthe steps of introducing the molecule with a composition to form amatrix; atomizing the matrix; removing the supernatant from the cells towhich the matrix is to be delivered; washing the cells; and contactingthe matrix with a plasma membrane by delivering 1 μL of matrix in theform of an aerosol to an area of 0.065-0.085 cm².

The method can include the further step of incubating the cells at roomtemperature for 0.1 seconds-2 minutes, optionally 2 minutes.

It has advantageously been found that the method can include theadditional step of contacting the cells with a second compositionincluding 68 mM NaCl, 1.4 mM KCl, 5 mM Na₂HPO₄, and 0.9 mM KH₂PO₄. Thesecond composition is a solution, optionally an aqueous solution havinga pH of 7.4. In a preferred embodiment, the additional contacting stepincludes delivering 1 μL of the second composition to an area of0.0052-0.0068 cm² for a period of 30 seconds at room temperature.

Following the additional contacting step, the method can further includethe step of exposing the cells to which the matrix is to be delivered,for example, by removing a substantial amount of the liquid surroundingthe cells by aspiration.

The method further includes culturing the cells after the exposing step,for example, by introducing suitable culture medium to the cells andincubating the cells in a humidified atmosphere with 5% CO2 at 37° C.

Accordingly, in a preferred embodiment, the method includes theadditional steps of contacting the cells with a second compositionincluding 68 mM NaCl, 1.4 mM KCl, 5 mM Na₂HPO₄, and 0.9 mM KH₂PO₄;exposing the cells to which the matrix is to be delivered; and culturingthe cells after the exposing step.

The present subject matter therefore also relates to a second aspect ofthe present subject matter, there is provided a second compositionincluding 68 mM NaCl, 1.4 mM KCl, 5 mM Na₂HPO₄, and 0.9 mM KH₂PO₄, whichcomposition can be an aqueous solution.

The present subject matter also relates to a method for deliveringmolecules of more than one molecular weight across a plasma membrane;the method including the steps of introducing the molecules of more thanone molecular weight to a composition to form a matrix; and contactingthe matrix with a plasma membrane.

Example Device for Delivery

The current subject matter further relates to delivering colloidalsuspension particles across plasma membranes, for example, bycontrolling colloidal droplet size. In particular, it has beendiscovered that intracellular delivery of materials can be achieved whena volume of an aqueous solution is contacted to a population of cells.The volume of aqueous solution that contacts the population can becontrolled, for example, by creating a controlled spray of the aqueoussolution. A colloidal suspension of the materials can be applied to cellmembranes using colloidal suspension droplets of a particular size (orrange of sizes). But when colloidal droplets are applied to a cellmembrane and colloidal droplet size is too large (and/or overall volumeis too great), damage to the cell may occur and cell viability isadversely affected. Conversely, when colloidal droplets are applied to acell membrane and the colloidal droplet size is too small (and/oroverall volume is too small), intracellular delivery of materials is notachieved. Therefore, control of colloidal droplet size (or production ofcolloidal droplets of or within a range of sizes) can enableintracellular delivery of materials. In some implementations, thepayload can be non-colloidal in size, e.g., less than 1 nanometer orgreater than 1000 nanometers in diameter.

Referring now to FIG. 1, there is shown a schematic diagram of anapparatus 10 for delivering a molecule across a plasma membraneaccording to an example implementation of the current subject matter.

Atomizers generate droplets when a sample (e.g., colloidal suspension ofdelivery material) is input under pressure, for example, using asyringe. The size of droplets produced can correlate to the amount ofpressure that is applied such that lower input pressure results inlarger droplet sizes. Because input pressure cannot be instantaneouslychanged, that is, it ramps (e.g., transitions) from zero or low pressureto a higher pressure, and likewise ramps (e.g., transitions) from ahigher pressure to a lower pressure, droplets produced have a wide rangeof sizes. A portion of the colloidal droplets produced can be too largefor a given intracellular delivery application. Because a portion of thecolloidal droplets produced are too large, cell death may occurnotwithstanding the production of appropriately sized colloidaldroplets. As described above, cell death is undesirable for someapplications. In addition, a portion of the colloidal droplets producedby atomizers can be too small, which leads to inefficient or ineffectiveintracellular delivery of materials.

The current subject matter enables production of colloidal droplets of aparticular size or range of sizes. In addition, the size of colloidaldroplets produced can be consistent, that is, production of dropletsoutside of the desired size or range of sizes is reduced and/orsubstantially eliminated. Control of colloidal droplet size can beachieved using a high-switching-speed valve with a cavity and/orensuring that there is sufficient headroom for an input air supply,which enables quick input pressure rise and falls times for an atomizer.The atomizer may be intended for use with a syringe.

What constitutes droplets that are too large and too small may varybased on application (e.g., materials to be delivered and type of targetcell). Therefore, intracellular delivery of materials can be achieved byproducing colloidal droplets and controlling the size of the colloidaldroplets. In some implementations, the colloidal droplets are producedin a manner so that substantially all colloidal droplets applied totarget cells have a size within a known/desired range that achievesintracellular delivery. In some implementations, formation of colloidaldroplets outside the known/desired range is minimized.

The apparatus 10 includes an atomizer 12 having at least one atomizeremitter 14; and a support 16 for supporting cells.

Contacting the matrix with a plasma membrane can include delivering thematrix in the form of an aerosol, which can be achieved using anatomizer.

The atomizer 12 can be selected from a mechanical atomizer, anultrasonic atomizer, an electrospray, a nebuliser, and a Venturi tube;and it is within the remit of the skilled person to select the atomizerbased on the requirements of delivering a molecule across a plasmamembrane. The atomizer 12 can be a commercially available atomizer, suchas a commercially available atomizer from LMA Teleflex of NC, USA.

The atomizer 12 is adapted to provide a colloid suspension of particles,each particle having a diameter of 30-100 μm. In certain embodiments,the atomizer 12 is adapted to provide a colloid suspension of particles,wherein each of the particles has a diameter of 50-80 μm. The particlesare liquid droplets including molecules to be delivered to the cells.

The atomizer 12 can include a gas reservoir 18. The apparatus 10 caninclude a pneumatic generator or gas reservoir 18 (also referred to as apneumatic generator). The gas in the gas reservoir 18 is maintainedunder pressure. The gas can be selected from air, carbon dioxide, andhelium; but it is understood that any suitable gas may be selected andused by the skilled person. The gas reservoir 18 can include a pressurehead generator, optionally a fixed pressure head generator to compressthe gas in the gas reservoir 18 and so maintain the gas under pressure.Examples of a gas reservoir 18 include bottled gases.

The gas reservoir 18 is in fluid communication with the atomizer emitter14. The gas reservoir 18 can be in fluid communication with the atomizeremitter 14, such that gas can flow from the gas reservoir 18 to theatomizer emitter 14. In certain embodiments, the gas reservoir 18includes a gas guide 20, which is in fluid communication with theatomizer emitter 14. Accordingly, the gas guide 20 is adapted to allowthe passage of gas therethrough. The gas guide 20 can be a hollow body,such as a hollow body having open ends. In an implementation, the gasguide 20 is a hollow body having first 22 and second 22′ open ends,optionally first 22 and second 22′ opposing open ends.

In an implementation, the diameter of the first 22 open end is differentto the diameter of the second 22′ open end. Preferably, the diameter ofthe first 22 open end is greater than the diameter of the second 22′open end. The first 22 open end can be in fluid communication with thegas reservoir 18. The second 22′ open end is preferably in fluidcommunication with the atomizer emitter 14. When a gas is injected underpressure from the gas reservoir 18 through the gas guide 20, thedecreasing section of the gas guide 20 resulting from the diameter ofthe first 22 open end being greater than the diameter of the second 22′open end, causes the speed of the gas flow to increase, therebygenerating a pressure drop at the second 22′ open end.

The apparatus 10 can further include a sample reservoir 24. The samplereservoir 24 is in fluid communication with the atomizer 12. In anexemplary implementation, the sample reservoir 24 is in fluidcommunication with the atomizer emitter 14. In preferred embodiments,the gas reservoir 18 and the sample reservoir 24 are both in fluidcommunication with the atomizer emitter 14. In such an arrangement,sample can be drawn from the sample reservoir 24 by the pressure drop atthe second 22′ open end of the gas guide 20. The sample can then beintroduced into the gas flow passing through the gas guide 20 from thegas reservoir 18 to the atomizer emitter 14.

In exemplary implementations, the apparatus 10 further includes a samplevalve 26 located between the sample reservoir 24 and the gas reservoir18. The sample valve 26 can be adapted to adjust the sample flow fromthe sample reservoir 24. The sample valve 26 can be used to allowcontinuous or semi-continuous sample flow. In an exemplaryimplementation, the sample valve 26 is adapted to allow semi-continuoussample flow of a defined amount of sample. For example, the sample valvecan be adapted to allow semi-continuous sample flow of 0.5-100 μL ofsample from the sample reservoir 24. In an exemplary implementation, thesample valve 26 is adapted to allow semi-continuous sample flow of 20 μLof sample from the sample reservoir 24. However, it is understood thatsample flow can be selected by a person skilled in the art, whereby thesample valve can be adapted to allow semi-continuous sample flow of 1 μLto an area of 0.065-0.085 cm².

The atomizer 12 and the support 16 are spaced apart. The support 16 canbe oriented toward the atomizer 12 such that the spray plume (sprayzone) generated by the atomizer 12 is received at or on the support 16.The support 16 includes a solid support. In some implementations, thesupport 16 includes a plate including sample wells. In alternativeembodiments, the support 16 includes a solid support for receiving andretaining a plate including sample wells. The support 16 or the platecan include sample wells selected from 1, 6, 9, 12, 24, 48, 96, 384, and1536 wells, for example, the support 16 or the plate can be a 1-, 6-,9-, 12-, 24-, 48-, 96-, 384-, or 1536-well plate. The support 16 can be,for example a biological membrane, such as a biological tissue, forexample a skin tissue or a tracheal tissue; or in some embodiments, abiological organ. The solid support can be formed from an inertmaterial.

In exemplary implementations, the solid support is formed from a plasticmaterial or a metal or metal alloy; although it is understood that anysuitable material may be selected and used by the skilled person. Thesupport 16 may be, in some embodiments, a synthetic membrane, such as analuminum membrane or a plastic membrane.

In exemplary implementations, the support 16 includes a heating element,which can be a resistive element, which can either increase or decreasethe temperature on or at the support 16.

The support 16 can be reciprocally mountable to the apparatus 10 toallow the support 16 to be reciprocally movable relative to theapparatus 10. In some implementations, the support 16 is reciprocallymovable relative to the atomizer 12 or the atomizer emitter 14. In suchan arrangement, the support 16 can be moved relative to the atomizeremitter 14 to achieve the optimal spray plume (spray zone) for deliveryof molecules across a plasma membrane. The support 16 can include asupport actuator to reciprocally move the support 16 relative to theatomizer 12 or the atomizer emitter 14, optionally the longitudinal axisof the atomizer emitter 14, thereby adjusting the distance between thesupport 16 and the atomizer emitter 14. The support 16 can additionallyinclude a support actuator to reciprocally move the support 16transverse to the longitudinal axis of the atomizer emitter 14, therebyadjusting the relative position of the support 16 and the atomizeremitter 14.

In an exemplary implementation, the distance between the atomizer 12 orthe atomizer emitter 14 and the support 16 is less than or equal to 31mm. The spaced apart atomizer 12 and support 16 define a spray zonethere between. In an implementation, the longitudinal length of thespray zone is 31 mm.

The longitudinal axis of the spray zone is preferably coaxial with thelongitudinal axis of the support 16. Additionally, the longitudinal axisof the atomizer emitter 14 is preferably coaxial with the longitudinalaxis of the support 16. In such an arrangement, the longitudinal axis ofthe atomizer emitter 14, the longitudinal axis of the support 16, andthe longitudinal axis of the spray zone are each coaxial, therebyensuring that the atomizer emitter 14, and the spray zone associatedwith the atomizer emitter 14, are centered over the support (forexample, the circular well of a plate) 16 for delivery.

The apparatus 10 can further include a valve 28 located between the gasreservoir 18 and the atomizer 12. The valve 28 can be anelectromagnetically operated valve, such as a solenoid valve.Alternatively, the valve 28 can be a pneumatic valve. The valve 28 ispreferably located at the gas guide 20 and can be adapted to adjust thegas flow within the gas guide 20. For example, the vale 28 can beswitchable between a closed position for preventing the gas fromactivating the atomizer 12 and an open position for allowing the gasunder pressure to activate the atomizer 12 to produce colloidaldroplets. The open position can be partially open so as to control thepressure that is received by the atomizer 12. The valve 28 can beadapted to allow continuous or semi-continuous gas flow. In an exampleimplementation, the valve 28 is adapted to allow semi-continuous gasflow of a defined time interval, for example, semi-continuous gas flowof a one second time interval.

The valve 28 can be adapted to allow continuous or semi-continuous gasflow. In a preferred embodiment, the valve 28 is adapted to allowsemi-continuous gas flow of a defined time interval, for example,semi-continuous gas flow of a one second time interval.

To ensure sterility and to remove foreign particles, the apparatus 10can further include at least one filter 30. In some implementations,each filter 30 has a pore size of less than 10 μm, but the skilledperson can readily determine the pore size to be used and selected. Eachfilter 30 is located at the gas guide 20, and each filter 30 is in fluidcommunication with the gas guide 20.

The apparatus 10 can include at least one regulator 32, which can be anelectrical regulator or a mechanical regulator. Each regulator 32 islocated at the gas guide 20 and is in fluid communication with the gasguide 20. Each regulator 32 can be a regulating valve and can be adaptedto adjust the pressure within the gas guide 20 to 1.0-2.0 bar. Eachregulating valve can also maintain the pressure within the gas guide 20at 1.0-2.0 bar. In exemplary implementations, each regulating valvemaintains the pressure within the gas guide 20 at 1.5 bar. Exemplaryimplementations of the current subject matter can include two regulators32. For example, the apparatus 10 can include first 32 and second 32′regulators. The first 32 and second 32′regulators are located at the gasguide 20 and are each in fluid communication with the gas guide 20. Inan exemplary implementation, the first regulator 32 is located betweenthe gas reservoir 18 and the filter 30. The first regulator 32 isadapted to adjust the pressure from the gas reservoir 18 within the gasguide 20 to 2.0 bar, and to maintain the pressure within the gas guide20 at 2.0 bar. The second regulator 32′ is located between the filter 30and the valve 28.

According some implementations, the atomizer emitter 14 is adapted toprovide a conical spray zone. The atomizer emitter 14 can be adapted toprovide a 30° conical spray zone.

The apparatus 10 can further include a microprocessor to control any orall parts of the apparatus 10; for example, the microprocessor can bearranged to control any or all of the sample valve 26, the supportactuator, the valve 28, and the regulator 32.

In some implementations, the apparatus 10 is arranged to deliver 1 μL ofsample to an area of 0.065-0.085 cm², optionally to deliver 1 μL ofmatrix to an area of 0.065-0.085 cm² of cells. The sample is can bedelivered in the form of an aerosol by atomizing the sample prior tocontacting the sample with a plasma membrane. The atomizer 12 can formdroplets of the sample, each droplet having a cross sectional dimensionof 30-100 μm, or more preferably, 50-80 μm. Optionally or additionally,the atomizer forms droplets of the sample, each droplet having a crosssectional dimension of less than 10 μm. The apparatus 10 is preferablyarranged such that delivery is conducted at a center point of the areato which the sample is to be delivered.

Materials and Methods.

All inorganic materials used were of ‘Analar’ grade, unless otherwisestated. All materials were of tissue culture grade and purchased fromSigma, unless otherwise stated.

100 ml of a first solution (Solution A) was prepared to a finalconcentration of 43 mM sucrose, 16 mM potassium chloride, 16 mM ammoniumacetate and 7 mM Hepes in molecular grade water, adjusted to pH 7.4 byadding 1.15 ml 1 M NaOH and filter sterilised using a filter with poresize 0.2 μm, before combining with ethanol in a ratio of 3:1.

100 ml of a second solution (Solution B) was prepared to a finalconcentration of 68 mM NaCl, 1.4 mM KCl, 5 mM Na₂HPO₄, and 0.9 mM KH₂PO₄in molecular grade water. The pH of the resultant solution was adjustedto pH 7.4 by adding 1.13 ml 1 M NaOH and was sterilised by autoclaving.

The molecules to be delivered to the cells included propidium iodide(668 Da), miRNA (15,000 Da) (Thermo Scientific), siRNA molecules (15,000Da) (Life Technologies), dextran (3000-2,000,000 Da) (LifeTechnologies).

Propidium iodide solution (1.0 mg/ml in water) was obtained from SigmaAldrich under Cat. No: P4864; non-targeting miRNA labelled with Dy547was obtained from ThermoScientific under Cat. No: CP-004500-01-05;fluorescein-labelled double stranded RNA oligomer (siRNA) was obtainedfrom Biosciences under Cat. No: 13750062; and fluorescein-labelleddextrans were obtained from Life Technologies Dextran 40,000 under Cat.No. D1845; Dextran 70,000 under Cat. No. D1823; Dextran 2,000,000 underCat. No. D7137; and Dextran 500,000 under Cat. No. D7139.

Molecules to be delivered to the cells were added to Solution A to forma matrix. The amount of molecules to be delivered was independent of theamount of molecules added to Solution A. In the present experiments, theamount of molecules added to Solution A was such that the matrixincluded 150 μM propidium iodide (668 Da), 3.3 or 6.6 μM miRNA (15,000Da), and 3.3 or 6.6 μM siRNA molecules (15,000 Da).

Cells and Cell Culture. T24 human bladder carcinoma, U373 glioblastoma,SKBR3 human hypertriploid, HeLa human epithelial adenocarcinoma, CHO-K1Chinese hamster ovary, COS-7 SV40 transformed kidney fibroblast, C2C12mouse myoblast; A549 adenocarcinomic human alveolar epithelial andBeas2B human bronchial epithelial cell lines were obtained from theAmerican Type Culture Collection (ATCC). HEK-n Human EpidermalKeratinocytes-neonatal and HDF Normal Human Dermal Fibroblasts celllines were obtained from Caltag MedSystems.

All cell lines were grown in a humidified atmosphere with 5% CO₂ at 37°C. Routine aseptic sub-culture of cells was carried out every 72 h orupon reaching 75-90% confluence, whichever occurred first.

For experiments, cells were seeded at a density of approximately 4×10³cells/well in 24-well plates and allowed to adhere for twenty four hourssuch that cells reached 75-90% confluence on the day of delivery.

Delivery of Molecules. An intranasal mucosal atomization devicecommercially available from LMA Teleflex of NC, USA under cataloguenumber MAD300 and including an atomizer emitter was set up as follows:the atomizer emitter was positioned 31 mm from the base of the 24-wellplate and above the center point of each circular well of the plate. Theatomizer emitter was adjusted to allow a pressure of 1.5 bar. A timerwas utilized to dispense spray from the atomizer emitter for a period of1 second. The atomizer emitter was primed by rinsing three times withSolution A containing molecules to be delivered.

Each well of the plate was treated as follows: cell culture medium wasremoved from the well using a micropipette. Optionally, the well wasrinsed twice with 250 μL phosphate buffered saline (PBS) using amicropipette. 20 μL of Solution A containing molecules to be deliveredwas delivered to the cells using the atomizer. The plate was incubatedat room temperature for a period of 30 seconds to 2 minutes depending onthe size of the molecule to be delivered, following which 250 μL ofSolution B was added to the well using a micropipette. The plate wasthen incubated at room temperature for 30 seconds, at which timeSolution B was removed from the well using a micropipette. 500 μL ofculture medium was added to the well using a micropipette. The cellswere then returned to a humidified atmosphere with 5% CO² at 37° C.

Fluorescence Microscopy. Fluorescein isothiocyanate (FITC)—and DyLightPhosphoramidite (DY547)-labelled molecules were used in accordance withthe manufacturer's instructions. Labelled molecules to be delivered tothe cells were added to Solution A and delivered to cells as describedabove herein. Following delivery, the plate was placed onto the stage ofa fluorescent microscope (Olympus CKX 41) and the cells were viewedusing filters to visualize fluorescence. Photomicrographs were acquired.

Flow Cytometry. Following delivery, cells were removed from each well ofthe plate using 200 μL trypsin. 200 μL culture medium was used toinactivate the trypsin. Cells were pelleted by centrifugation for 5minutes at 259 relative centrifugal force (RCF) and the pellet wasre-suspended in 200 μL PBS using a micropipette tip. The cell suspensionwas loaded into a flow cytometer [Accuri Flow Cytometyer, BDBiosciences] and the fluorescence was analyzed according tomanufacturer's instruction.

Cell Viability. Following delivery, cell viability was assessed usingthe CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS)(Promega) according to the manufacturer's instructions. In short, mediumwas removed from the well by aspiration and replaced with 200 μL freshmedium to which 40 μL MTS reagent was added. The plate was incubated at37 degrees C. for 1 hour protected from light. 100 μL of solution wasremoved from the well and placed into a 96-well plate and absorbance wasread at 450 nm using a GloMax 96 Microplate luminometer [Promega].

Co-localization visualization. Fluorescein isothiocyanate (FITC)—andDyLight Phosphoramidite (DY547)-labelled molecules were used inaccordance with the manufacturer's instructions. Following delivery, theplate was placed onto the stage of a fluorescent microscope [Olympus CKX41] and the cells were viewed using filters to visualize fluorescence.Photomicrographs were acquired.

The current subject matter further relates to delivering colloidalsuspension particles across plasma membranes, for example, bycontrolling colloidal droplet size. In particular, it has beendiscovered that intracellular delivery of materials can be achieved whena colloidal suspension of the materials is applied to cell membranesusing colloidal suspension droplets of a particular size (or range ofsizes). But when colloidal droplets are applied to a cell membrane andcolloidal droplet size is too large, damage to the cell may occur andcell viability is adversely affected. Conversely, when colloidaldroplets are applied to a cell membrane and the colloidal droplet sizeis too small, intracellular delivery of materials is not achieved.Therefore, control of colloidal droplet size (or production of colloidaldroplets of or within a range of sizes) can enable intracellulardelivery of materials.

Atomizers generate droplets when a sample (e.g., colloidal suspension ofdelivery material) is input under pressure, for example, using asyringe. The size of droplets produced can correlate to the amount ofpressure that is applied such that lower input pressure results inlarger droplet sizes. Because input pressure cannot be instantaneouslychanged, that is, it ramps (e.g., transitions) from zero or low pressureto a higher pressure, and likewise ramps (e.g., transitions) from ahigher pressure to a lower pressure, droplets produced have a wide rangeof sizes. A portion of the colloidal droplets produced can be too largefor a given intracellular delivery application. Because a portion of thecolloidal droplets produced are too large, cell death may occurnotwithstanding the production of appropriately sized colloidaldroplets. As described above, cell death is undesirable for someapplications. In addition, a portion of the colloidal droplets producedby atomizers can be too small, which leads to inefficient or ineffectiveintracellular delivery of materials.

The current subject matter enables production of colloidal droplets of aparticular size or range of sizes. In addition, the size of colloidaldroplets produced can be consistent, that is, production of dropletsoutside of the desired size or range of sizes is reduced and/orsubstantially eliminated. Control of colloidal droplet size can beachieved using a high-switching-speed valve with a cavity and/orensuring that there is sufficient headroom for an input air supply,which enables quick input pressure rise and falls times for an atomizer.The atomizer may be intended for use with a syringe.

What constitutes droplets that are too large and too small may varybased on application (e.g., materials to be delivered and type of targetcell). Therefore, intracellular delivery of materials can be achieved byproducing colloidal droplets and controlling the size of the colloidaldroplets. In some implementations, the colloidal droplets are producedin a manner so that substantially all colloidal droplets applied totarget cells have a size within a known/desired range that achievesintracellular delivery. In some implementations, formation of colloidaldroplets outside the known/desired range is minimized.

The apparatus 10 can further include a valve 28 located between the gasreservoir 18 and the atomizer 12. The valve 28 can be anelectromagnetically operated valve, such as a solenoid valve. The valve28 can be a pneumatic valve. The valve 28 can be located at the gasguide 20 and can be adapted to adjust the gas flow within the gas guide20. For example, the vale 28 can be switchable between a closed positionfor preventing the gas from activating the atomizer 12 and an openposition for allowing the gas under pressure to activate the atomizer 12to produce colloidal droplets. The open position can be partially openso as to control the pressure that is received by the atomizer 12. Thevalve 28 can be adapted to allow continuous or semi-continuous gas flow.In an example implementation, the valve 28 is adapted to allowsemi-continuous gas flow of a defined time interval, for example,semi-continuous gas flow of a one second time interval.

In some implementations, the switching speed of the valve 28 can be lessthan 250 milliseconds. The switching speed can be the time required forthe valve 28 to transition between the closed position and open position(and/or vice versa). In some implementations, the valve 28 has aswitching speed that is less than 200 milliseconds. In someimplementations, the valve 28 has a switching speed between 50 and 200milliseconds. Other implementations are possible.

The valve 28 can include a cavity.

In some implementations, the atomizer 12 can produce colloidal dropletshaving a diameter between 30 and 100 micrometres. In someimplementations, the atomizer 12 can produce colloidal droplets having adiameter between 30 and 50 micrometres. In some implementations, becauseof the characteristics of apparatus 10 (e.g., such as a fast valve 26switching time), the pressure that inputs to the atomizer 12 results ingreater than 80 percent of the colloidal droplets produced by theatomizer 12 as having a diameter between 30 and 100 micrometres (asmeasured over a 1 second period in which the valve transitions at leastonce from the closed position to the open position or from the openposition to the closed position). In some implementations, the pressurethat inputs to the atomizer 12 results in greater than 99 percent of thecolloidal droplets produced by the atomizer 12 as having a diameterbetween 30 and 100 micrometres (as measured over a 1 second period inwhich the valve transitions at least once from the closed position tothe open position or from the open position to the closed position).

In operation, the current subject matter can enable intracellulardelivery of molecules. FIG. 3 is a process flow diagram illustrating aprocess 800 of producing colloidal droplets for delivering a sample tothe cytoplasm of one or more target cells. At 810, a gas can begenerated by a pneumatic generator or gas reservoir 18. The gas can beunder pressure. At 820, a valve can be switched from a closed positionfor preventing the gas under pressure from activating an atomizer 12 andan open position for allowing the gas under pressure to activate theatomizer to produce colloidal droplets. The valve 28 can be between thepneumatic generator or gas reservoir 8 and the atomizer 12. A sample canbe provided from a sample reservoir for the atomizer to producecolloidal droplets. Other implementations are possible.

Additional example implementations follow.

Example 1: Development of Technique

Delivery of molecules into living cells is highly desirable for a widerange of applications. Generally, the types of molecules involved can becategorised according to the mass of the molecule: (i) small chemicalmolecules generally have an average molecular weight of: <1,000 Da; (ii)peptides generally have an average molecular weight of: ˜5,000 Da; (iii)siRNA molecules generally have an average molecular weight of: ˜15,000Da; (iv) antibodies generally have an average molecular weight of:˜150,000 Da; and (v) nucleic acids, such as DNA, generally have anaverage molecular weight of: ˜5,000,000 Da.

A variety of approaches are taken to deliver molecules across a plasmamembrane and into a cell, each approach depending on the size andchemistry of molecule to be delivered. Organic solvents, such as DMSO,have been used to deliver small chemical molecules. While the molecularbasis of the action of DMSO on a plasma membrane is still obscure, DMSOis known to exhibit three distinct modes of action, each over adifferent concentration range. At low concentrations, DMSO inducesplasma membrane thinning and increases fluidity of the hydrophobic coreof the plasma membrane. At higher concentrations, DMSO induces transientwater pores in the plasma membrane. At still higher concentrations,individual lipid molecules are irreversibly desorbed from the plasmamembrane followed by a detrimental disintegration of the bilayerstructure of the plasma membrane.

Introduction of larger, biological molecules such as oligopeptides,polypeptides or proteins, and nucleic acids (such as plasmid DNA,oligonucleotides, and siRNA) is referred to as ‘transfection’. Use oftraditional delivery compositions, such as DMSO, are not efficient fordelivery of these larger molecules. siRNA molecules are usuallydelivered by liposome-mediated transfection (lipofection). Plasmid DNAis usually delivered using biological (viruses), chemical (lipid-basedor chemical polymers), or physical (electroporation, magnetofection,injection) methods. However, these methods are not well-suited forproteins and peptides, and furthermore, many cell types, particularlyprimary cells and stem cells, remain ‘hard to transfect’ even withnucleic acid molecules where high toxicity levels are often a problem.

A wide range of methods are also used to chemically ‘permeabilise’ cellsand tissues. However, the vast majority of these methods are not aimedat ‘reversible permeabilisation’ and delivery into a living cell.Instead, the methods are usually aimed at ‘irreversiblepermeabilisation’ to deliver a ‘label’ that will attach to a molecule orstructure within a cell or tissue for purposes such as visualisation orquantification (for example, immunofluorescence). In these situations,the cells and tissues are non-viable following permeabilisation.Chemicals typically used in these methods include alcohols (whichdissolve lipids in a plasma membrane), detergents (which create pores ina plasma membrane) and enzymes (which digest proteins and create poresin a plasma membrane).

A small number of studies have reported successful reversiblepermeabilisation using detergents. Detergents (e.g., surfactants) arewidely used in biology for protein extraction from cell membranes and asmembrane permeabilizing agents. Triton X-100 (TX100) is one of the mostwidely used non-ionic surfactants for lysing cells or to permeabilizethe living cell membrane for transfection. Other exemplary surfactantsinclude polysorbae 20 (e.g., Tween 20),3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), sodium dodecyl sulfate (SDS), and octyl glucoside. However,cell viability is extremely sensitive within a narrow range ofsurfactant concentrations and controlling the TX100 concentration fortransfection is difficult. Van den Ven et al. report using TX100 todeliver molecules ranging from 1,000 MW to 150,000 MW to cultured cells(van de Ven K., et al., J. Biomed Opt 2009:14(2), incorporated herein byreference in its entirety). Medepalli et al. report using saponin inconjunction with a hypotonic buffer (sucrose, KCl, potassium acetate,Hepes) to deliver nanometer sized quantum dots to cultured cells(Medepalli, K., et al., Nanotechnology 2013; 24:20, incorporated hereinby reference in its entirety). This hypotonic buffer is used to supportcell viability by providing ions and pH buffering to the cells whilstalso being hypotonic with the intention that water should flow into thepermeabilised cells and bring the payload with it (note that water isitself toxic to cells). However, the experiments of the van de Ven andMedepalli reports have been unable to be repeated.

A vector-free delivery method was developed based on reversiblepermeabilisation that would facilitate delivery of payloads into cellsin a manner that would retain cell viability and payload functionality.As other groups have done, the following hypothesis was utilized:firstly, permeabilisation could be induced by chemical modification ofthe cell membrane; secondly, delivery could be enhanced via osmoticpressure brought about by using a hypotonic delivery solution wherebyinflux of water into the permeabilised cells facilitated influx of apayload and thirdly, cell survival could be enhanced if the hypotonicdelivery solution was also buffered and physiological. Based on initialobservations, further hypotheses were developed and refined as describedlater here. For chemical permeabilisation, the most commonpermeabilising agents are detergents which interact with certaincomponents in cell membranes to create holes (Hapala, I., Crit Rev.Biotech. 1997; 17(2): 105-22). Medepalli et al reported delivery ofquantum dots into cultured cells by incubating cells in a specifichypotonic physiological buffered solution termed ‘S Buffer’ (78 mMsucrose, 30 mM KCl, 30 mM potassium acetate, 12 mM HEPES) for 5 min at4° C. (Medepalli K. et al., Nanotechnology 2013; 24(20)). In someexamples, potassium acetate is replaced with ammonium acetate in the “S”buffer. They also stated that delivery could be enhanced by addingsaponin to the solution. However, high levels of cell damage anddetachment of A549 cells were observed under these conditions and didnot observe uptake of labelled siRNA and dextran molecules. Organicsolvents such as alcohols can permeabilise cells by dissolving lipidfrom the cell membrane. A reversible permeabilising protocol usingethanol as the permeabilising agent was made.

A range of ethanol concentrations in several diluents including waterand PBS as well as various concentrations of S Buffer were examined.Replacing potassium acetate in the S buffer with ammonium acetate gavebetter delivery efficiencies, because of effects on the cell membrane. Apreferred delivery solution composition which gave desirable initialresults was 75% H₂O, 25% ethanol, 32 mM sucrose, 12 mM KCl, 12 mMammonium acetate and 5 mM Hepes and was used from this point on unlessotherwise stated. However, that this solution induced significanttoxicity when pipetted directly onto cells in large volumes, therebysoaking or submerging the cells, FIG. 56A. When 200 μl delivery solution(per well of a 24-well plate) containing PI was pipetted directly ontoexposed cells, most cells immediately stained positive for PI (FIG. 17).LDH release measured at 24 hr post-delivery indicated that approximately50% cells were damaged (FIG. 19). In contrast, no delivery of largermolecules such as 10-kDa dextran-Alexa488 or siRNA-FITC was observed(FIG. 17). The cells were being over-permeabilised to the point oflethal damage where PI could enter and bind to nuclear DNA but osmoticpressure gradients could not be established to deliver the largerpayloads.

Therefore, to minimize damage, the delivery process can be as rapid aspossible with the maximum volume of payload being delivered in thesmallest volume and shortest time practicable. Cells were seeded into24-well plates on Day 0 such that they were 80-90% confluent on Day 1when delivery was carried out. Supernatant was removed from the targetwell and 20 μl delivery solution containing PI, 10-kDa dextran-Alexa488or siRNA-FITC was pipetted into the well. Cells were incubated for 2 minat room temperature (RT) to facilitate uptake. To further facilitateuptake and prevent cell dehydration, 200 μl 0.5×-PBS was then added andcells were incubated for a further 30 sec. This solution was thenremoved and 400 μl culture medium was added. The cells were thenanalyzed by fluorescence microscopy. With this method, PI uptake wasapparent at the edge of the well but not in the center (FIG. 18).Delivery results were also inconsistent with this method. No delivery of10-kDa dextran-Alexa488 or siRNA-FITC was observed (FIG. 17 and FIG.18). LDH levels were reduced however compared with the larger 200 μlvolume (FIG. 19). Over-permeabilisation of some cells andunder-permeabilisation of others was taking place. Simultaneous deliveryof the permeabilising solution to all cells was preferable to ‘droppingon’ volumes using a micropipette where not all cells were targeted atthe same time. Furthermore, the volume reaching a cell should besufficient to permit influx of water into the cell but insufficient tobring the cell to bursting point. In other words, the volume should betitered to match the absorbance capacity of the cells. A spray-mediateddelivery achieved these outcomes, whereby the spray maximized contact ofthe payload with the cell membrane of the target cells in a very shorttimeframe and in a uniform manner, resulting in preservation of cellviability and reliable and robust uptake of payload across the cellmembrane and into the interior of the cells (FIG. 56B).

Instrument. To implement this approach, an instrument was configuredincluding x, y and z (FIG. 20). The instrument was used to deliver10-kDa dextran to A549 cells. Cells were seeded into 48-well plates inorder to match the cell monolayer area to the spray diameter.Supernatant was removed from the target well and 10 μl delivery solutioncontaining 10-kDa dextran was sprayed onto the cells. Following a 2 minincubation at RT, 200 μl 0.5×-PBS was added and cells were incubated fora further 30 sec. This solution was then removed and 400 μl culturemedium was added. This method resulted in successful delivery of 10-kDadextran into cells with efficiencies of greater than 50% and little tono toxicity compared with untreated cells (FIG. 21 and FIG. 22).

Having established a technique for reversibly permeabilising cells, thetime taken for recovery of the cells was examined. Delivery solution wassprayed in the absence of payload and propidium iodide (PI) wassubsequently added to the culture medium at time points up to 1 hourpost-spray in order to detect permeabilised cells. While PI uptake wasvisible at 5 min post-spray, the number of PI-positive cells wassubstantially reduced by 30 min and 60 min post-spray, as illustrated inFIG. 23.

Example Optimal Parameters.

Several parameters were optimized in the course of developing thetechnique. The distance of the sprayhead from the cells, the pressure ofthe spray, the volume of delivery solution sprayed per well and theconcentration of ethanol were fine tuned to maximize delivery efficiencywhile minimizing toxicity (FIGS. 25-29). A distance of 31 mm between thesprayhead and the cells, a spray pressure of 1.5 bar, a volume of 10 μlfor 48-well plates and an ethanol concentration of 25% were theparameters that produced optimal delivery efficiencies and toxicitylevels.

Example 2

Effect of Delivering a Molecule Having an Average Molecular Weight of Upto 15,000 Da Across a Plasma Membrane According to the Present SubjectMatter.

In this example, a FITC-labelled siRNA molecule having an averagemolecular weight of 15,000 Da was delivered to cells using an apparatusaccording to the present subject matter. The siRNA molecules wereintroduced to a composition, which was an aqueous solution including 32mM sucrose, 12 mM KCl, 12 mM ammonium acetate, 5 mM hepes, a pH of about7.4, 20, 30, or 40% (v/v) of ethanol; and 6.6 μM molecules to bedelivered; in order to form a matrix. 1 μL of matrix was delivered to anarea of 0.065-0.085 cm², such that the matrix was contacted with theplasma membrane of the cells either directly using a micropipette orusing an apparatus as described herein. The relative amount of moleculesdelivered (the amount of fluorescence) and the cell viability (amount ofviable cells) was assessed and expressed as a percentage. The resultsare illustrated in FIG. 4.

As is illustrated in FIG. 4, delivery of a molecule having an averagemolecular weight of up to 15,000 Da using a method of the presentsubject matter (black bars) increases the delivery rate of the molecule(e.g., percent of cells showing successful delivery of the molecule)compared to delivery of the molecule by contacting with the plasmamembrane of the cells directly using a micropipette (hashed lines).Indeed, in a composition including 30 or 40% (v/v) of ethanol, anddelivery of the resultant matrix directly using a micropipette, nodelivery of molecules was detected in viable cells.

Example 3

Effect of Delivering a Molecule Having an Average Molecular Weight of Upto 1,000 Da Across a Plasma Membrane According to the Current SubjectMatter

In this example, a propidium iodide molecule having an average molecularweight of 668 Da was delivered to cells using a method according to thepresent subject matter. The molecules were introduced to a composition,which was an aqueous solution including 32 mM sucrose, 12 mM KCl, 12 mMammonium acetate, 5 mM hepes, a pH of about 7.4, 20 or 40% (v/v) ofethanol; and 150 μM molecules to be delivered; in order to form amatrix. 1 μL of matrix was delivered to an area of 0.065-0.085 cm², suchthat the matrix was contacted with the plasma membrane of the cellseither directly using a micropipette or an apparatus as described aboveherein. The results are illustrated in FIG. 4.

As is illustrated in FIG. 5, delivery of a molecule having an averagemolecular weight of up to 668 Da in a matrix using a method according tothe present subject matter (black bars) increases the delivery rate ofthe molecule (e.g., percent of cells showing successful delivery of themolecule), (y-axis shows percent delivered, and x-axis shows percentethanol) compared to delivery of the molecule by contacting with theplasma membrane of the cells directly using a micropipette (hashedlines). Indeed, in a composition including 40% (v/v) of ethanol, anddelivery of the resultant matrix directly using a micropipette, nodelivery of molecules was detected in viable cells.

Example 4

Delivering molecules of more than one molecular weight across a plasmamembrane. In this example, a first molecule of propidium iodide havingan average molecular weight of 668 Da and a second molecule ofFITC-labelled dextran having a molecular weight of 40,000 were bothsimultaneously delivered to cells using an apparatus of the presentsubject matter. The first and second molecules were simultaneouslyintroduced to a composition, which was an aqueous solution including 32mM sucrose, 12 mM KCl, 12 mM ammonium acetate, 5 mM hepes; a pH of about7.4, 25% (v/v) of ethanol; and 150 μM molecules to be delivered; inorder to form a matrix. 1 μL of matrix was delivered to an area of0.065-0.085 cm², such that the matrix was contacted with the plasmamembrane of the cells in the form of an aerosol using the method of thepresent subject matter. The results are illustrated in FIGS. 6A-6C.

As is illustrated in FIG. 6A, delivery of a first molecule having anaverage molecular weight of 668 Da in a matrix using the present subjectmatter results in delivery of the molecule into the cell. FIG. 6Billustrates simultaneous delivery of a second molecule having an averagemolecular weight of 40,000 Da in the same matrix using the presentsubject matter results in simultaneous delivery of the molecule into thecell. The simultaneous delivery is illustrated in FIG. 6C.

Example 5

Effect of Delivering a Molecule Having an Average Molecular Weight of Upto 500,000 Da Across a Plasma Membrane According to the Present SubjectMatter

In this example, a molecule of FITC-labelled dextran having an averagemolecular weight of 10,000 Da was delivered to cells using an apparatusaccording to the present subject matter. The molecules were introducedto a composition, which was an aqueous solution including 32 mM sucrose,12 mM KCl, 12 mM ammonium acetate, 5 mM hepes; a pH of about 7.4; 25%(v/v) of ethanol; and 150 μM molecules to be delivered; in order to forma matrix. 1 μL of matrix was delivered to an area of 0.065-0.085 cm²,such that the matrix was contacted with the plasma membrane of the cellseither directly using a micropipette or an apparatus of the presentsubject matter. The results are illustrated in FIG. 7.

As is illustrated in FIG. 7, delivery of a molecule having an averagemolecular weight of up to 500,000 Da in a matrix using a method of thepresent subject matter (black bar) increases the delivery rate of themolecule (e.g., percent of cells showing successful delivery of themolecule), (the y-axis shows percent delivered, and x-axis indicatespercent ethanol) compared to delivery of the molecule by contacting withthe plasma membrane of the cells directly using a micropipette (hashedlines). Indeed, in a composition including 25% (v/v) of ethanol, anddelivery of the resultant matrix directly using a micropipette, nodelivery of molecules was detected in viable cells.

Example 6

Effect of Contacting Cells with a Second Composition Including 68 mMNaCl, 1.4 mM KCl, 5 mM Na₂HPO4, and 0.9 mM KH₂PO₄.

In this example, a FITC-labelled siRNA molecule having an averagemolecular weight of 15,000 Da was delivered to cells using an apparatusaccording to the present subject matter. Following delivery of moleculesto cells, cells were contacted with 200 μL of a second compositionincluding one of: Dulbecco's Modified Eagle's Medium (DMEM) with fetalbovine serum (FBS); DMEM without FBS; distilled water (H₂O); an aqueoussolution of 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄(1×PBS); an aqueous solution of 68 mM NaCl, 1.4 mM KCl, 5 mM Na₂HPO₄,and 0.9 mM KH₂PO₄ (0.5×PBS); or an aqueous solution of 13.7 mM NaCl, 0.3mM KCl, 1.0 mM Na₂HPO₄, and 0.18 mM KH₂PO₄ (1×PBS) for 30 seconds beforeaddition of culture medium and assessment of delivery using fluorescencemicroscopy as described herein. The results are shown in FIG. 8, whichillustrates.

As illustrated in FIG. 8, an aqueous solution of 68 mM NaCl, 1.4 mM KCl,5 mM Na₂HPO₄, and 0.9 mM KH₂PO₄ (0.5×PBS) is preferred to maintain cellviability in a method according to the present subject matter (y-axisindicates percent delivered).

Example 7

Delivering Molecules of Different Molecular Weight Across a PlasmaMembrane

In this example, molecules of propridium iodide (668 Da), FITC-labelledsiRNA (15,000 Da), Dy547-labelled miRNA (15,000 Da), FITC-labelleddextran (40,000 Da), and FITC-labelled dextran (500,000 Da) were eachdelivered to cells using an apparatus according to the present subjectmatter. The molecules were each separately introduced to a composition,which was an aqueous solution including 32 mM sucrose, 12 mM KCl, 12 mMammonium acetate, 5 mM hepes; a pH of about 7.4, wherein the compositionincluded 25% (v/v) of ethanol; and 150 μM molecules to be delivered; inorder to form a matrix. 1 μL of each matrix (each containing a differentmolecule to be delivered) was delivered to an area of 0.065-0.085 cm²,such that the matrix was contacted with the plasma membrane of the cellseither directly using a micropipette or by a method according to thepresent subject matter. Cells were visualized at 0 hour (propidiumiodide) or at 24 hours post-delivery (siRNA-FITC, miRNA-Dy547 anddextran-FITC). Photomicrographs showing (A) fluorescence and (B) phasecontrast were obtained using an Olympus IX71 Inverted Microscope. Theresults are illustrated in FIG. 9.

As illustrated in FIG. 9, molecules of varying molecular weights can besuccessfully delivered to cells using an apparatus according to thepresent subject matter. Additionally, varying molecular weights ofdextran (e.g., 3 kDa, 40 kDa, 70 kDa, 500 kDa, and 2,000 kDa), andproteins (e.g., beta-lactoglobulin, HRP, ovalbumin, BSA, catalase, andapoferritin) can be successfully delivered, as shown in FIGS. 29 and 43,respectively.

The present subject matter therefore can provide an apparatus fordelivering a molecule across a plasma membrane, and which enables thedelivery of molecules to living cells by reversible permeabilisation ofthe or each cell. Reversible permeabilisation allows each cell to bepermeable, optionally temporarily permeable, thereby allowing uptake ofmolecules into the cell. Advantageously, permeability can be reversedbefore unacceptably high levels of cell death occur.

Example 8

Effect of Solute Content on the Delivery of a Molecule Having an AverageMolecular Weight of Up to 15,000 Da.

In this example, an siRNA molecule having an average molecular weight of15,000 Da was delivered to cells as generally described herein above.The composition used was an aqueous solution having a pH of about 7.4,wherein the composition included 25% (v/v) of ethanol; and 3.3 μMmolecules to be delivered. A 1× solution was prepared by adding sucrose,KCl, ammonium acetate, and hepes to a final concentration of 32 mMsucrose, 12 mM KCl, 12 mM ammonium acetate, and 5 mM hepes. Further testsolutions were prepared with varying solute (sucrose, KCl, ammoniumacetate, and hepes) concentrations of 0.2×, 0.4×, 0.6×, 0.8×, 2×, 2.4×,and 2.8×. The results are illustrated in FIG. 10.

As is illustrated in FIG. 9, for delivery of a molecule having anaverage molecular weight of up to 15,000 Da, a composition including asolute concentration of sucrose, KCl, ammonium acetate, and hepes of1×-2×, optionally 1× is preferred (black bars), given that cell toxicity(white bars) is minimal at these concentrations. This equates to asolute concentration of 32-64 mM sucrose, 12-24 mM KCl, 12-24 mMammonium acetate, and 5-10 mM hepes; further optionally 32 mM sucrose,12 mM KCl, 12 mM ammonium acetate, and 5 mM hepes.

Example 9

Effect of Alcohol Concentration on the Delivery of a Molecule Having anAverage Molecular Weight of Up to 15,000 Da

In this example, an siRNA molecule having an average molecular weight of15,000 Da was delivered to cells as generally described herein above.The composition used was an aqueous solution including 32 mM sucrose, 12mM KCl, 12 mM ammonium acetate, and 5 mM hepes, a pH of about 7.4, 6.6μM molecules to be delivered, and 5, 10, 20, 30, and 40% (v/v) ofethanol. The results are illustrated in FIG. 11.

As is illustrated in FIG. 11, for delivery of a molecule having anaverage molecular weight of up to 15,000 Da, a composition including2-45% (v/v) of the alcohol, optionally 20-30% (v/v) of the alcohol,further optionally 25% (v/v) of the alcohol is preferred (black bars)while minimizing cell toxicity (white bars).

As a comparative test, an siRNA molecule having an average molecularweight of 15,000 Da was delivered to cells as generally described hereinabove, wherein the composition used was an aqueous solution including 32mM sucrose, 12 mM KCl, 12 mM ammonium acetate, 5 mM hepes, a pH of about7.4, 6.6 μM molecules to be delivered, and 5, 10, 20, and 30% (v/v) ofmethanol. The results are illustrated in FIG. 12.

As is illustrated in FIG. 12, for delivery of a molecule having anaverage molecular weight of up to 15,000 Da, a composition including2-45% (v/v) of the alcohol, optionally 10-20% (v/v) of the alcohol,further optionally 20% (v/v) of the alcohol is preferred (black bars)while minimizing cell viability (white bars).

Example 10

Effect of Salt Content on the Delivery of a Molecule Having an AverageMolecular Weight of Up to 1,000 Da

In this example, a propridium iodide molecule having an averagemolecular weight of 668 Da was delivered to cells as generally describedherein above. The composition used was an aqueous solution having a pHof about 7.4, wherein the composition included 25% (v/v) of ethanol; 150μM molecules to be delivered. The test solutions were prepared with 25%of 0.5×, 1×, 2×, and 4× phosphate buffered saline (PBS), which equatesto a salt content of 19.0 mM, 37.9 mM, 75.8 mM, and 151.6 mM. Theresults are illustrated in FIG. 12.

Example 11

Effect of Alcohol Concentration on the Delivery of a Molecule Having anAverage Molecular Weight of Up to 1,000 Da

In this example, a propridium iodide molecule having an averagemolecular weight of 668 Da was delivered to cells as generally describedherein above. The composition used was an aqueous solution including 32mM sucrose, 12 mM KCl, 12 mM ammonium acetate, 5 mM hepes, a pH of about7.4, 150 μM molecules to be delivered, and 5, 10, 20, 30, and 40% (v/v)of ethanol. The results are illustrated in FIG. 14.

As is illustrated in FIG. 14, for delivery of a molecule having anaverage molecular weight of up to 1,000 Da, a composition including2-45% (v/v) of the alcohol, optionally 20-30% (v/v) of the alcohol,further optionally 25% (v/v) of the alcohol is preferred.

As a comparative test, a propridium iodide molecule having an averagemolecular weight of 668 Da was delivered to cells as generally describedherein above, wherein the composition used was an aqueous solutionincluding 32 mM sucrose, 12 mM KCl, 12 mM ammonium acetate, 5 mM hepes,a pH of about 7.4, 150 μM molecules to be delivered, and 5, 10, 20, and30% (v/v) of methanol. The results are illustrated in FIG. 14.

As is illustrated in FIG. 15, for delivery of a molecule having anaverage molecular weight of up to 1,000 Da, a composition including5-20% (v/v) of the alcohol, optionally 5, 10, or 20% (v/v) of thealcohol is preferred (black bars) while minimising cell toxicity (whitebars).

As a further comparative test, a propridium iodide molecule having anaverage molecular weight of 668 Da was delivered to cells as generallydescribed herein above, wherein the composition used was an aqueoussolution including 32 mM sucrose, 12 mM KCl, 12 mM ammonium acetate, 5mM hepes, a pH of about 7.4, 150 μM molecules to be delivered, and 2%(v/v) of butanol. The results are illustrated in FIG. 16.

As is illustrated in FIG. 16, for delivery of a molecule having anaverage molecular weight of up to 5,000 Da, a composition including 2%(v/v) of butanol is preferred (black bars) while minimizing celltoxicity (white bars).

The present subject matter therefore provides a method for delivering amolecule across a plasma membrane, and which enables the delivery ofmolecules to living cells by reversible permeabilization of the cells oreach cell. Reversible permeabilization allows the cells or each cell tobe permeable, optionally temporarily permeable, thereby allowing uptakeof molecules into the cell. Advantageously, permeability can be reversedbefore unacceptably high levels of cell death occur.

Example 12

Effect of delivering a molecule having an average molecular weight of upto 40,000 Da across a plasma membrane according to the current subjectmatter.

In this example, a molecule of FITC-labelled dextran having an averagemolecular weight of 40,000 Da was delivered to cells using an apparatusaccording to the current subject matter. The molecules were introducedto a composition, which was an aqueous solution including 32 mM sucrose,12 mM KCl, 12 mM ammonium acetate, 5 mM hepes; a pH of about 7.4; 40%(v/v) of ethanol; and 10 μM molecules to be delivered; in order to forma matrix. 1 μL of matrix was delivered to an area of 0.065-0.085 cm²,such that the matrix was contacted with the plasma membrane of the cellseither directly using a micropipette or an apparatus of the currentsubject matter.

As is illustrated in FIG. 7, delivery of a molecule having an averagemolecular weight of up to 40,000 Da in a matrix using the presentsubject matter (black bars) increases the delivery rate of the moleculecompared to delivery of the molecule by contacting with the plasmamembrane of the cells directly using a micropipette (hashed lines).Indeed, in a composition including 40% (v/v) of ethanol, and delivery ofthe resultant matrix directly using a micropipette, no delivery ofmolecules was detected in viable cells.

Example 13

Effect of Delivering Molecules with a Range of Molecule Types and Sizes

In this example, the ability of the spraying method to addresschallenges in delivery of a broad range of molecule types and sizes wereexamined. Dextrans of increasing sizes, including 3 kDa, 40 kDa, 70 kDa,500 kDa and 2,000 kDa were successfully delivered into A549 cells, asillustrated in FIG. 29. Other types of molecules with various dimensionssuch as linear siRNA molecules (approximately 15 kDa) and large antibodymolecules (150 kDa) were also delivered, as illustrated in FIG. 30.Moreover, different types of molecules were delivered in a wide varietyof combinations. For example, DAPI, phallotoxin and MitoTracker Red weresuccessfully co-delivered into A549 cells, as was the combination of10-kDa dextran-Alexa488 and DAPI, as illustrated in FIG. 31.

Because spraying is a vectorless delivery method, of particular note isthe ability to deliver mRNA and plasmid DNA with this approach. ReportermRNAs encoding green fluorescent protein (GFP) and luciferase weresprayfected into CHO cells. GFP expression was observed by fluorescencemicroscopy and luciferase expression was detected by luminometry wascomparable with Lipofectamine 2000 controls (FIG. 33 and FIG. 34).Similarly, DNA plasmids encoding GFP and luciferase were expressed whensprayfected into CHO cells (FIG. 35 and FIG. 36). These data demonstratethe functionality of nucleic acid payloads following delivery intocells. Furthermore, the ability to address adherent cells, and with verylow toxicity, is important for primary and stem cell populations wherelarge numbers of cells may not be available and minimal manipulation andpassaging steps are desirable.

Example 14

Effect of Delivery Across Cell Types, Including Adherent Cell Lines,Primary Fibroblasts, Primary Stem Cells and Suspension Cells.

In this example, the delivery method across cell types was evaluated.The delivery technique was successfully deployed across a wide range ofadherent cell types including A549 and CHO cell lines as well as primaryfibroblasts, as shown in FIG. 37 and primary MSC, shown in FIG. 39.Furthermore, the protocol was successfully adapted to address suspensioncells such as U226 human multiple myeloma cells, shown in FIG. 41A. Thecell suspension was placed into a porous cell culture plate insert and abrief gentle vacuum of approximately −0.5 to −0.68 bar was applied for20-45 sec to remove supernatant before the cells were sprayed (FIG. 41Aand FIG. 42).

Additionally, the protocol was successfully adapted to addresssuspension cells such as Jurkat cells, T-lymphocyte cells, shown in FIG.41B. DAPI and Mitotracker Red were successfully delivered to the Jurkatcells (FIG. 41B top and middle panel, respectively). Furthermore, mRNAencoding for GFP was delivered to Jurkat cells, and GFP expression wasobserved at 24 hours post-delivery.

Example 15

Evaluation of the Delivery Technology on the Intracellular Delivery ofProteins.

A notable application of delivery technology is the intracellulardelivery of proteins. Proteins are a very diverse group in terms oftheir size, shape and chemistry and few methods are currently availablefor efficient delivery of these molecules. A broad range of proteins ofincreasing sizes from 18.3 kDa to 443 kDa were labeled with either FITCor Alexa-488 and their delivery by spraying was examined. All proteinswere successfully delivered (FIG. 43 and FIG. 44) into CHO cells. Ageneral trend towards declining delivery efficiencies with increasingsize of protein (FIG. 44) was observed. To further confirm that proteinswere delivered into cells, ovalbumin-FITC was delivered and subsequentlydetected by immunofluorescence using an anti-ovalbumin antibody (FIG.45). For a given protein, in this case beta-lactoglobulin, a doseresponse was evident with increasing efficiency of delivery evident withincreasing concentration of protein sprayed (FIG. 46).

Example 16

Evaluation of the Functionality of Proteins Post-Delivery into Cells.

The functionality of proteins post-delivery into cells was examined.Various assays are available for the detection of horse radishperoxidase (HRP) activity and two assays were used to detect HRPactivity following spraying into CHO cells. Firstly, the Tyramide SignalAmplification (TSA™) assay was adapted, which normally uses thecatalytic activity of HRP to generate high density labelling of a targetprotein or nucleic acid sequence in situ. The Alexa Fluor® 488-labelledtyramide substrate was used to demonstrate activity and localization ofHRP in CHO cells following delivery by spraying (FIG. 47). Secondly, aDCFH-DA assay was used to quantify HRP activity. 2′,7′dichlorofluorescin diacetate (DCFH-DA) is a hydrophobic non-fluorescentmolecule that penetrates rapidly into cells and is hydrolyzed byintracellular esterases to give the DCFH molecule which can be oxidizedto its fluorescent product 2′,7′dichlorofluorescein (DCF) which can bemeasured. HRP was sprayfected into CHO cells and the cells wereincubated with DCFH-DA. Increasing production of DCF was observed withincreasing dose of HRP delivered (FIG. 48). No toxicity was observedwith this assay (FIG. 49).

Example 17

Labeling Primary MSC by Spraying for Tracking to Target Organs wasEvaluated.

Several cell types, including MSC, are used for in vivo cell therapyapplications. However, success with many of these strategies has beenhampered by lack of understanding about cell trafficking in the body.The efficiency of trafficking to target organs versus sequestration innon-target organs is difficult to investigate and delivery of labeledcells in animal studies is often used to understand these processes.Efficient and rapid labeling of cells is not currently achievable.Standard fluorescent labels such as FITC and other fluorophores areusually not bright enough to be detected in situ in tissues and animals.Brighter labels such as quantum-dots (Q-dots) have been more recentlydeveloped but these require extended periods of incubation with cells,usually overnight, in order to achieve satisfactory levels of labelling.The method of the current subject matter is a rapid delivery methodwhereby payloads are delivered within minutes to target cells. Theability of the method to deliver Q-dots to primary MSC was examined, andwhether these could be detected in situ following ex vivo injection inmouse spleens.

Q-dots were sprayfected into cultured primary mouse MSC, as illustratedin FIG. 50. Spleens were dissected from mice and 2×10⁵ sprayfected MSCin 100 μl culture medium were injected into the spleens. Fluorescence inthe spleens was examined by 3-dimensional cryoimaging using the Cryovisinstrument. Q-dots were detected in the spleens as shown in FIG. 51.

Example 18

Experimentally Measured Volume Delivered Per Cell in A549 Cells, CHOCells, and MSCs.

The areas of three different cell lines (A549, CHO, and MCSs) wereexperimentally calculated and measured (FIG. 55). The average area foreach of the cell lines was measured to be 932 μm², 372 μm², and 2054 μm²for A549, CHO, and MCSs, respectively. Thus, the calculated number ofcells per well (based on the size of a 48-well cell culture plate), wascalculated to be 102,500, 255,000, and 46200 for A549, CHO, and MCSs,respectively. Upon delivery of 10 μL, approximately 9.8×10⁻⁵ μL per cellwere delivered to A549 cells, 3.9×10⁻⁵ μL per cell were delivered to CHOcells, and 2.2×10⁻⁴ μL per cell were delivered to MSCs. Theexperimentally measured volume delivered per cell of these threeexamples fall within the range of the theoretical calculations (e.g.,6.0×10⁻⁷ microliter per cell and 7.4×10⁻⁴ microliter per cell) utilizingcell diameter estimations from the ATCC, Celeromics Technologies, andother cell culture references known by one skilled in the art.

In the descriptions above and in the claims, phrases such as “at leastone of” or “one or more of” may occur followed by a conjunctive list ofelements or features. The term “and/or” may also occur in a list of twoor more elements or features. Unless otherwise implicitly or explicitlycontradicted by the context in which it is used, such a phrase isintended to mean any of the listed elements or features individually orany of the recited elements or features in combination with any of theother recited elements or features. For example, the phrases “at leastone of A and B;” “one or more of A and B;” and “A and/or B” are eachintended to mean “A alone, B alone, or A and B together.” A similarinterpretation is also intended for lists including three or more items.For example, the phrases “at least one of A, B, and C;” “one or more ofA, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, Balone, C alone, A and B together, A and C together, B and C together, orA and B and C together.” In addition, use of the term “based on,” aboveand in the claims is intended to mean, “based at least in part on,” suchthat an unrecited feature or element is also permissible.

The subject matter described herein can be embodied in systems,apparatus, methods, and/or articles depending on the desiredconfiguration. The implementations set forth in the foregoingdescription do not represent all implementations consistent with thesubject matter described herein. Instead, they are merely some examplesconsistent with aspects related to the described subject matter.Although a few variations have been described in detail above, othermodifications or additions are possible. In particular, further featuresand/or variations can be provided in addition to those set forth herein.For example, the implementations described above can be directed tovarious combinations and subcombinations of the disclosed featuresand/or combinations and subcombinations of several further featuresdisclosed above. In addition, the logic flows depicted in theaccompanying figures and/or described herein do not necessarily requirethe particular order shown, or sequential order, to achieve desirableresults. Other implementations may be within the scope of the followingclaims.

What is claimed is:
 1. A method for delivering a payload across a plasmamembrane of a cell, comprising: providing a population of cells; andcontacting the population of cells with a volume of aqueous solution,whereby the payload is subsequently delivered across the plasma membraneof the cell, the aqueous solution including the payload and an alcoholat a concentration greater than 2 percent (v/v); wherein the populationof cells is in contact with the aqueous solution for 2 seconds to 2minutes prior to adding a second volume of buffer or culture medium tosubmerse or suspend the population of cells, wherein the population ofcells comprises non-adherent cells, and the non-adherent cells includeat least one of primary or immortalized hematopoietic stem cell (HSC), Tcells, natural killer (NK) cells, cytokine-induced killer (CIK) cells,human cord blood CD34+ cells, B cells, or induced pluripotent stemcells; wherein contacting the population of cells with the volume ofaqueous solution is performed by gas propelling the aqueous solution toform a spray.
 2. The method of claim 1, wherein the volume of aqueoussolution including the payload is between 6.0×10⁻⁷ microliter per celland 7.4×10⁻⁴ microliter per cell.
 3. The method of claim 1, furthercomprising creating a cell occupied area by at least removing media fromthe population of cells such that the non-adherent cells rest on thecell occupied area, wherein a total volume of aqueous solution isdelivered to the cell-occupied area, wherein (a) the total volume ofaqueous solution is 20 μl and the cell-occupied area is about 1.9 cm²,wherein about is within 10 percent; or (b) the total volume of aqueoussolution is 10 μl and the cell-occupied area is about 0.95 cm², whereinabout is within 10 percent.
 4. The method of claim 1, wherein saidaqueous solution comprises an ethanol concentration of 5 to 30% (v/v).5. The method of claim 1, wherein said aqueous solution comprises one ormore of 75 to 98% H₂O, 2 to 45% ethanol, 6 to 91 mM sucrose, 2 to 35 mMpotassium chloride, 2 to 35 mM ammonium acetate, and 1 to 14 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES).
 6. Themethod of claim 1, wherein the payload comprises a small chemicalmolecule, a peptide or protein, or a nucleic acid.
 7. The method ofclaim 6, wherein (a) the small chemical molecule comprises a molecularmass of less than 1,000 Da; (b) wherein the small chemical moleculecomprises MitoTracker® Red CMXRos, propidium iodide, methotrexate, orDAPI (4′,6-diamidino-2-phenylindole); (c) the peptide comprisesecallantide, liraglutide, or icatibant; (d) the nucleic acid comprises asmall-interfering RNA (siRNA), and wherein the siRNA molecule comprisesa molecular mass of about 15,000 Da; (e) the protein comprises amolecular mass about 1,000-150,000 Da; (f) the protein comprises anantibody, or fragment thereof, and wherein the antibody or fragmentthereof comprises an anti-actin antibody, an anti-GAPDH antibody, ananti-Src antibody, an anti-Myc ab, and an anti-Raf antibody; or (g) thenucleic acid molecule comprises greater than 5,000,000 Da.
 8. The methodof claim 1, wherein the payload comprises a therapeutic agent, adiagnostic agent, a fluorescent molecule, or a detectable nanoparticle.9. The method of claim 8, wherein (a) the therapeutic agent includes atleast one of cisplatin, aspirin, a statin, and fluoxetine; (b) thediagnostic agent includes at least one of methylene blue, patent blue V,and indocyanine green; or (c) the nanoparticle comprises a quantum dot.10. The method of claim 1, further comprising creating a cell occupiedarea by at least removing media from the population of cells such thatthe non-adherent cells rest on the cell occupied area, wherein thepopulation of cells at rest on the cell occupied area is substantiallyconfluent, wherein substantially is greater than 75 percent confluent.11. The method of claim 1, further comprising creating a cell occupiedarea by at least removing media from the population of cells such thatthe non-adherent cells rest on the cell occupied area, wherein thepopulation of cells at rest on the cell occupied area form a monolayerof cells.
 12. The method of claim 1, wherein the population of cellscomprises macrophages.
 13. The method of claim 1, wherein the populationof cells comprises mesenchymal stem cells.
 14. The method of claim 1,wherein the payload comprises nucleic acid.
 15. The method of claim 1,wherein the payload comprises mRNA.
 16. The method of claim 1, whereinthe aqueous solution includes the alcohol at a concentration greaterthan 5 percent (v/v).
 17. The method of claim 1, wherein said aqueoussolution comprises an ethanol at a concentration of 2 to 30% (v/v). 18.The method of claim 1, wherein the volume of aqueous solution isdelivered to the population of cells in the form of the spray.
 19. Themethod of claim 18, wherein the spray comprises a colloidal suspensioncomprising a diameter of 1 nm to 100 μm.
 20. The method of claim 1,wherein the buffer or culture medium comprises a second aqueoussolution.
 21. The method of claim 20, wherein the second aqueoussolution comprises saline.
 22. The method of claim 21, wherein thesaline comprises phosphate buffered saline (PBS).
 23. A method fordelivering a payload across a plasma membrane of a cell, comprising:providing a population of cells; and contacting the population of cellswith a volume of aqueous solution, whereby the payload is subsequentlydelivered across the plasma membrane of the cell, the aqueous solutionincluding the payload and an alcohol at a concentration greater than 2percent (v/v), wherein the population of cells is in contact with theaqueous solution for 2 seconds to 2 minutes prior to adding a secondvolume of buffer or culture medium to submerse or suspend the populationof cells, wherein the population of cells comprises adherent cells, andthe adherent cells include at least one of primary or immortalizedmesenchymal stem cells, lung cells, neuronal cells, fibroblasts, humanumbilical vein (HUVEC) cells, and human embryonic kidney (HEK) cells,wherein contacting the population of cells with the volume of aqueoussolution is performed by gas propelling the aqueous solution to form aspray.
 24. The method of claim 23, wherein said aqueous solutioncomprises an ethanol concentration of 5 to 30% (v/v).
 25. A method fordelivering a payload across a plasma membrane of a cell, comprising:providing a population of cells; and contacting the population of cellswith a volume of aqueous solution, whereby the payload is subsequentlydelivered across the plasma membrane of the cell, the aqueous solutionincluding the payload and an alcohol at a concentration greater than 2percent (v/v), wherein the population of cells is in contact with theaqueous solution for 2 seconds to 2 minutes prior to adding a secondvolume of buffer or culture medium to submerse or suspend the populationof cells, wherein contacting the population of cells with the volume ofaqueous solution is performed by gas propelling the aqueous solution toform a spray, and wherein the buffer or culture medium comprises asecond aqueous solution.
 26. The method of claim 25, wherein thepopulation of cells comprises adherent cells, and the adherent cellsinclude at least one of primary or immortalized mesenchymal stem cells,lung cells, neuronal cells, fibroblasts, human umbilical vein (HUVEC)cells, or human embryonic kidney (HEK) cells.
 27. The method of claim25, wherein the population of cells comprises non-adherent cells, andthe non-adherent cells include at least one of primary or immortalizedhematopoietic stem cell (HSC), T cells, natural killer (NK) cells,cytokine-induced killer (CIK) cells, human cord blood CD34+ cells, or Bcells.
 28. The method of claim 25, wherein said aqueous solutioncomprises an ethanol concentration of 5 to 30% (v/v).